Lectin compositions and methods for modulating an immune response to an antigen

ABSTRACT

The present invention relates to a fusion polypeptide comprising at least about 10 contiguous amino acid residues of an influenza virus hemagglutinin and at least about 5 contiguous amino acids of a naturally occurring GM-CSF molecule.

BACKGROUND OF THE INVENTION

[0001] Typically, the specific modulation of an immune response to anantigen in a subject requires the administration of another substance,e.g. an adjuvant, in admixture with the antigen in order to initiateand/or direct the modulation. Traditional adjuvants, though, have anumber of weaknesses. For example, many are crude, heterogeneouspreparations. In addition, many are relatively weak immunomodulators,and some cause severe local inflammation that is unacceptable in humans.Purified soluble polypeptides, such as cytokines, have some advantagesover crude adjuvants, but their value is limited because they diffuseaway from the antigen upon administration. While certain cell-surfacemolecules may be potential immunomdulators as components of cell-basedvaccines, their application generally involves gene transfer into thecells, which is often problematic.

[0002] The invention therefore fills heretofore unmet needs by providingmolecules that can bind to antigen bearing targets, such as cells,viruses, and isolated antigens, and that can serve as immunomodulatorswhen administered with an antigen bearing target. In addition, theinvention provides compositions comprising these molecules and relatedmethods. The compositions and methods of the invention are also usefulfor other applications, e.g. any application in which it is desirable toattach a biological effeector, such as a polypeptide ligand for a cellsurface receptor, to a target structure, such as a virus or a cell.

SUMMARY OF THE INVENTION

[0003] The present invention relates to a multifunctional molecule, e.g.a fusion polypeptide, comprising a first part which is capable ofbinding to an antigen bearing target and a second part which is capableof binding to a cell. In a preferred embodiment, the first part is afirst cell surface binding moiety and the second part is a second cellsurface binding moiety. In this embodiment, the first cell surfacebinding moiety can attach to a virus or cell, e.g. a tumor cell, whichcomprises an antigen. It is also particularly preferred that the secondcell surface binding moiety can bind to a cell-surface polypeptide, e.g.a non-immunoglobulin polypeptide, of an antigen presenting cell (APC).Thus, in some embodiments, a multifunctional molecule of the inventioncan serve as a bridge or link between an antigen bearing target and anAPC.

[0004] As used herein an “antigen bearing target” is an entity whichcomprises an antigen. As used herein an “antigen bearing target”includes, for example, a whole cell which expresses an antigen a cellfraction comprising an antigen, a membrane fraction comprising anantigen, a virus comprising an antigen, a viral particle comprising anantigen, or an antigen, e.g. a polypeptide antigen, which may be free ofany other cell-derived or virus-derived material. Cellular fractions maybe prepared using methods known to those of skill in the art such asthose taught in Cell Biology A Laboratory Handbook (Academic Press 1994Editor J. E. Celis ISBN 0-12-164715-3)

[0005] The term “antigen” as used herein refers to a molecule againstwhich a subject can initiate a humoral and/or cellular immune response.Antigens can be any type of biologic molecule including, for example,simple intermediary metabolites, sugars, lipids, and hormones as well asmacromolecules such as complex carbohydrates, phospholipids, nucleicacids and proteins. Common categories of antigens include, but are notlimited to, viral antigens, bacterial antigens, fungal antigens,protozoa and other parasitic antigens, tumor antigens, antigens involvedin autoimmune disease, allergy and graft rejection, and othermiscellaneous antigens. In the compositions and methods of theinvention, it is preferred that the antigen is a polypeptide, e.g., onecomprising at least seven amino acids.

[0006] As used herein, “antigen presenting cell” or “APC” refers tocells that ingest and present antigen to T cells. These cells includephagocytic leukocytes, macrophages, and dendritic cells, B lymphocytes,and endothelial cells. A “professional APC” is an APC that isconstitutively able to activate a T lymphocyte. Professional APCstypically constitutively express class II major histocompatibilitymolecules and costimulatory molecules such as B7-1 and/or B7-2.

[0007] In one embodiment, the invention encompasses a multifunctionalmolecule, the first part of which is a lectin. Thus, the multifunctionalmolecule can bind to one or more carbohydrates of an antigen bearingtarget. In a preferred embodiment of the invention, the first part ofthe multifunctional molecule is a lectin and the second portion is aligand for a cell surface receptor. Preferably, the cell surfacereceptor is a cell surface receptor of an APC. Ligands for a cellsurface receptor include any ligand which will bind to a cell surfacereceptor, and preferably include, but are not limited to, an opsonin,cytokine, adhesion molecule, counterreceptor of a T cell costimulatorymolecule, a defensin, a ligand for a CD40 molecule, or a heat shockprotein, or a portion of any of these ligands, including about (or atleast about) 5, 8, 10, 12, 15, 20, 25, 35, 50, 60, 70, 80, 100, or 120contiguous amino acids of such a ligand. Preferably, the multifunctionalmolecule which comprises first and second parts comprises an amino acidsequence which can bind to a cell surface receptor including, but notlimited to an adhesion molecule, a costimulatory molecule for a T cell,or a receptor for at least one of the following types of molecules: acytokine, a defensin, a heat shock protein, a CD40 molecule, or anopsonin.

[0008] A cell surface receptor useful in the present invention is anycell surface molecule which can bind the ligand portion of amultifunctional molecule of the invention. Preferably, the cell surfacereceptor is a CD40 molecule, a T cell costimulatory molecule, anadhesion molecule, or a receptor for a cytokine, a defensin, a heatshock protein, an opsonin, or an adhesion molecule. Cell surfacereceptors, useful in the invention include, but are not limited to thecell surface molecules identified by GenBank Accession number inAppendix I and II, or those cell surface molecules which are encoded bya nucleic acid molecule identified by GenBank Accession number inAppendix I or II.

[0009] The term “cytokine” as used herein refers to a polypeptidemolecule that is naturally secreted by mammalian cells and that binds toa cell surface receptor on a leukocyte, inducing a change (e.g., achange in the proliferative state, a change in the transcriptionalprofile or a change in the propensity to migrate) in the leukocyte(other than mere occupancy of the leukocyte's receptors for thecytokine). “Change” refers to at least about a 5% increase or decreaseas compared to in the absence of a cytokine. The term “cytokine” alsorefers herein to a polypeptide molecule that is a ligand for a receptorfor a naturally occurring cytokine.

[0010] Examples of cytokines which are useful in the methods andcompositions of the invention include the following: GM-CSF, Il-2, IL-4,IL-6, IL-12, ligands for hematopoietin receptors, ligands forimmunoglobulin superfamily receptors, ligands for interferon receptors,ligands for TNF receptors, and ligands for chemokine receptors. Anantibody against a cytokine receptor can also be a cytokine.

[0011] In one embodiment of the invention, it is preferred that acytokine comprised by a composition of the invention promote a Th1immune response, i.e., the generation of T cells that express Th1cytokines such as IL-2 and IFN-γ. In another embodiment, it is preferredthat a cytokine comprised by a composition of the invention promote aTh2 immune response, i.e., the generation of T cells that express Th2cytokines such as IL-4 and IL-10.

[0012] “Engineered cytokines” as described herein are cytokines whichcomprise a heterologous cell surface binding moiety.

[0013] The term “opsonin” as used herein refers to naturally occurringand non-naturally occurring molecules which are capable, by virtue ofbeing contemporaneously bound or attached to both an antigen-containingcell and an antigen-presenting cell (APC), of acting as a link orcoupling agent (an adapter) between the antigen and the APC to allowmore efficient binding, engulfment, and internalization of theantigen-containing cell by the APC. An opsonin useful according to theinvention, also includes non-naturally occurring opsonins capable ofbinding to APCs via receptors that can bind naturally occurringopsonins.

[0014] The term “opsonin” as used herein can also refer to moleculeswhich can be processed such that at least one product of the processingstep or steps is capable of, by virtue of being contemporaneously boundor attached to both an antigen-containing cell and an APC, acting as alink or coupling agent to allow more efficient binding, engulfment, andinternalization of other antigen-containing cells by the APC. An opsonincan also be any polypeptide chain of a multichain opsonin.

[0015] Examples of opsonins which are useful in the methods andcompositions of the invention include the following: vitronectin,fibronectin, complement components such as C1q (including any of itscomponent polypeptide chains A, B and C), complement fragments such asC3d, C3b and C4b, mannose binding protein, conglutinin, surfactantproteins A and D, C-reactive protein (CRP), alpha-2-macroglobulin, andimmunoglobulins, for example, the Fc portion of an immunoglobulin.

[0016] “Innate opsonins” are opsonins of the innate immune system andare known in the art as secreted polypeptide molecules of the innateimmune system and are believed to bind contemporaneously to an antigenand to the surface of an APC. They can thus act as “bridges”, and arethought, by virtue of this property, to promote internalization ofantigens by APCs. The mode in which opsonins bind to antigens variesamong opsonins, and can be covalent or noncovalent. In general, theantigen-binding moieties of innate opsonins differ from theantigen-binding moieties of immunoglobulins in that the former arerelatively invariant among members of the same species, and do notundergo diversification during the ontogeny of an individual.

[0017] A molecule containing a naturally occurring APC-binding moietyshall be considered an opsonin if it contains a moiety through which itcan be stably bound or attached to a cell such that the APC-bindingmoiety is located in the extracellular space, whether or not the opsoninmolecule contains its natural antigen-binding domain.

[0018] “Engineered opsonins”, as described herein, include molecules inwhich a cell surface binding moiety is substituted for the naturalantigen-binding domain of an opsonin or where a cell surface bindingmoiety is linked to the opsonin without modification or removal of thenatural antigen-binding domain of the opsonin.

[0019] A “cell surface binding moiety” is a moiety through which amolecule can be stably bound to a cell surface, e.g. a cell wall, apolysaccharide capsule, or the lipid or protein component of a plasmamembrane, or to the surface of a virus. Such moieties include but arenot limited to crosslinking moieties and lipid moieties. It is preferredthat the cell surface binding moiety bind to a cell by a means otherthan interaction of a polypeptide with its cognate cell-surfacepolypeptide. It is further preferred that the cell surface bindingmoiety comprise a non-polypeptide moiety. In a preferred embodiment, alipid moiety is linked to the engineered molecule via aglycosylphosphatidylinositol (GPI) moiety. In another preferredembodiment, the lipid comprises a fatty acid, e.g. palmitate. In yetanother preferred embodiment of the invention, the cell surface bindingmoiety is linked to an opsonin or an antigen-binding domain-truncatedopsonin at the antigen-binding end of the opsonin. In another preferredembodiment, the multifunctional molecule comprises an idiotypic portionof an immunoglobulin which can bind to an APC. Preferably, the opsoninof an opsonin-enhanced cell is one of alpha' chain C3b or mannosebinding protein.

[0020] If the opsonin is a fragment of C3, it is preferred hat it bindto CR1 with a greater affinity than to CR2. It is further preferred thatthe fragment of C3 not be a ligand for CR2. Preferably, the opsonin isneither C3bi, C3d, nor C3dg.

[0021] It is preferred that the opsonins bind to receptors that triggerphagocytosis and that are non-clonotypic and thus do not vary from cellto cell as, for example, clonotypic receptors do. Non-clonotypicreceptors are present on cells which play a role in innate immunity, andinclude, e.g., non-idiotypic receptors. Examples of such receptorsinclude CR1, CR2, CR3, CR4, and C1q receptor, receptors containing acomponent of the C1q receptor, collectin receptors, receptors for α2 m,receptors for CRP, and Fc receptors for immunoglobulins.

[0022] “Exogenous” refers to something which is introduced from orproduced outside the cell.

[0023] “Endogenous” refers to something which is expressed or presentnaturally in a cell.

[0024] “Heterologous” refers to something which is not naturallyexpressed in a cell.

[0025] Preferably, the multifunctional molecule which comprises firstand second parts can bind, via the second part, to the surface or plasmamembrane of an antigen presenting cell (APC), i.e. a cell that canpresent antigen to a T cell, e.g. a cell that can activate a T cell, atleast in part by presenting antigen to the T cell. The APC may be aleukocyte, e.g. a cell of monocytic lineage and/or a dendritic cell.Preferably, binding of the multifunctional molecule is independent ofexpression of an idiotype, e.g. a clonotypic determinant of animmunoglobulin, on the APC. Most preferably, the multifunctionalmolecule comprises a first end which can bind to a cell that comprisesan antigen and a second end which can bind to a APC.

[0026] The multifunctional molecule may bind to an antigen bearingtarget cell by, e.g., inserting into the lipid portion of a cellmembrane or by binding to a structure, e.g. a polypeptide or acarbohydrate, that is physically associated with the lipid portion ofthe membrane. The structure need not be directly in contact with thelipid portion of the membrane, but may be indirectly attached, e.g. acarbohydrate that is part of a cell-surface glycoprotein. Preferably themultifunctional molecule can bind via a first part to an antigen bearingtarget, preferably a mammalian cell that comprises an antigen, and via asecond part to an APC. The invention also encompasses the use of amolecule that can bind via a first part to a virus or to a non-mammaliancell, e.g. a fungal or bacterial cell, and via a second part to an APC.In the latter cases, the first part may bind, e.g., to a component of acell wall or a capsule.

[0027] In a preferred embodiment, the multifunctional molecule whichcomprises first and second parts comprises a first part which comprisesa lectin and a second part that can bind to a leukocyte, e.g. an APC,e.g. a cell of monocytic lineage or a dendritic cell (which may itselfbe of monocytic lineage). A “lectin”, according to the invention, is amolecule or part of a molecule, e.g. an amino acid sequence, which canbind to a carbohydrate, e.g. a polysaccharide. Families of naturallyoccurring lectins include:

[0028] 1) Galectins, a rapidly growing family of animal lectins. All ofthem share galactose-specificity.

[0029] 2) Calcium-dependent (C-type) animal lectins, an extremely largefamily composed of members having diverse structures and functions.

[0030] 3) Among this C-type lectin family, selectins form adistinguishable subfamily by their specific function in leukocyteadhesion to endothelial cells through sialyl-LewisX recognition.

[0031] 4) Collectins, another subfamily of C-type lectins specific formannose, which have a unique structure consisting of a C-type lectindomain and a collagen-like domain. They are involved in innate immunity.

[0032] 5) Invertebrates are known to contain various lectins in theirbody fluids, probably as body-protection factors. Recently, some lectinsfrom an echinoderm were found to show hemolytic activity.

[0033] 6) Annexins, a group of proteins having affinity to lipids thatwere recently shown to be lectins showing binding to glycosaminoglycans.

[0034] 7) The legume lectin family, which consists of a large number ofmembers, such as ConA, with variable saccharide specificity comparableto C-type lectins.

[0035] 8) Ricin, the first lectin investigated in Russia more than 100years ago. It is now evident that the ricin family has many otherhomologous members which differ in either toxicity or sugar-bindingspecificities.

[0036] Thus, a multifunctional molecule of the invention may bind to oneor more carbohydrates. Carbohydrates to which lectins may bind alsoinclude, for example, carbohydrates comprising lactose, D-mannose,D-glucose, D-fucose, L-fucose (e.g. alpha-L-fucose), D-galactose, bloodgroup A oligosaccharides, blood group B oligosaccharides, saccharidescomprisingalpha-D-Gal(1->3)[alpha-Lfuc(1->2)]-beta-D-Gal(1->¾-beta-D-GlcNAc,saccharides comprising alpha-sialyl [2->3]-lactose, alpha-D-mannosylglycoconjugates, alpha-NeuNAc-[2->6]-Gal, alpha-NeuNAc-[2->6]-GalNAc,alpha-NeuNAc-[2->3]-Gal, N-acetyl-terminal alpha-D-galactosyl residues,terminal beta-D-galactosyl residues, N-acetyllactosamine, terminalalpha-D-mannosyl residues, N-acetyl-beta-D-glucosamine, terminalN-acetyl-D-galactosamine, N-acetylneuraminic acid, and terminalalpha-D-galactosaminyl residues.

[0037] The multifunctional molecule which comprises a lectin maycomprise, for example, the whole of a naturally occurring lectin or aportion of a naturally occurring lectin, e.g about (or at least about)5, 8, 10, 12, 15, 20, 25, 35, 50, 60, 70, 80, 100, or 120 contiguousamino acids of a naturally occurring polypeptide lectin. In oneembodiment the multifunctional molecule comprises a carbohydrate-bindingdomain of a naturally occurring lectin, i.e., a portion of a lectin thatcan bind to a carbohydrate in the absence of the remainder of thelectin. In another embodiment the lectin may be non-naturally occurring,e.g. identified from an artificial library of molecules or designed bymodifying the structure of a naturally occurring lectin.

[0038] Lectins known as “hemagglutinins” bind to carbohydrates onerythrocytes, e.g. blood group antigens, and when incubated with thesecells cause them to aggregate. The influenza virus hemagglutinin, forexample, binds to sialic acid (as does the human parainfluenza virus 3hemagglutinin/neuraminidase). There are at least 15 known influenzahemagglutinin subtypes, defined by their distinct antigenic properties.Any of these subtypes, designated, e.g., H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, and H15, may provide amino acid sequencesuseful in the compositions and methods of the invention. In oneembodiment of the invention, the hemagglutinin is of a subtype from avirus that infects humans, e.g. H1, H2, or H3. In another embodiment,the hemagglutinin is of a subtype from a virus that does not infecthumans, e.g. one of H4 through H15. Amino acid sequences can vary up toabout 20% for influenza hemagglutinins within a given subtype, and canvary between about 30% and about 70% for influenza hemagglutinins fromdifferent subtypes.

[0039] Influenza hemagglutinin is expressed as a single polypeptidechain, designated HA0, which trimerizes post-translationally. HA0 isproteolytically cleaved to yield two domains, HA1 and HA2, which aredisulfide-bonded to each other. HA1 comprises significant sialic acidbinding activity, while HA2 is anchored to the viral membrane andfacilitates fusion of this membrane with a host cell membrane. Inpreferred embodiments of the invention, the multifunctional moleculecomprising first and second parts comprises an amino acid sequence of anHA1 domain.

[0040] The molecule may be a fusion polypeptide which comprises one ormore amino acids interposed between the first and second parts whichbind to cells, e.g. a fusion polypeptide which comprises a first aminoacid sequence which can bind to an antigen bearing target and a secondamino acid sequence which can bind to a leukocyte, and which furthercomprises at least one amino acid interposed between the first andsecond parts. The interposed amino acids may comprise, e.g., a linkersequence intended to lessen steric hindrance or other undesirableinteractions between the aforementioned first and second parts. For,example, one such type of sequence takes the form (Gly₃Ser)_(n).Additional useful linkers include, but are not limited to(Arg—Ala—Arg—Asp—Pro—Arg—Val—Pro—Val—Ala—Thr)₁₋₅ (Xu et al., 1999, Proc.Natl. Acad. Sci. U.S.A. 96: 151-156), (Gly—Ser)_(n)(Shao et al., 2000,Bioconjug. Chem. 11: 822-826), (Thr—Ser—Pro)_(n)(Kroon et al., 2000,Eur. J. Biochem. 267: 6740-6752), (Gly—Gly—Gly)_(n)(Kluczyk et al.,2000, Peptides 21: 1411-1420), and (Glu—Lys)_(n)(Klyczyk et al., 2000,supra), wherein n is 1 to 15 (each of the preceding references is alsoincorporated herein by reference). In another embodiment, no amino acidsare interposed between the first and second parts.

[0041] The present invention further provides a nucleic acid molecule,preferably a recombinant nucleic acid molecule which encodes amultifunctional polypeptide of the present invention. The nucleic acidmolecule may be,for example, DNA, RNA, cDNA, or mRNA. The nucleic acidmolecule may be naturally occuring or may be partially or whollysynthesized using techniques known to those of skill in the art. In apreferred embodiment, the nucleic acid molecule is a DNA moleculecomprising a first nucleic acid sequence encoding a first amino acidsequence which can bind to an antigen bearing target, and a secondnucleic acid sequence encoding a second amino acid sequence which canbind to a cell surface receptor on an APC.

[0042] The present invention still further provides a vector comprisingthe nucleic acid molecule encoding a multifunctional polypeptide of theinvention, e.g. an expression vector suitable for expressing in a hostcell, wherein the host cell is preferably a eukaryotic cell, morepreferably an animal cell, more preferably a mammalian cell, and stillmore preferably a human cell. In another preferred embodiment the hostcell is a yeast cell, e.g. Saccharomyces cerevesiae.

[0043] The invention also provides a host cell comprising a nucleic acidvector which comprises a sequence encoding the multifunctional moleculeof the present invention. Preferably, the host cell is a eukaryoticcell, such as a yeast cell- or an animal cell, preferably a human cell.The host cell may also be a prokaryotic cell.

[0044] The invention also encompasses a molecule, e.g. a polypeptide,e.g. a fusion polypeptide, which comprises a first part that can bind toan antigen bearing target, e.g. a cell, e.g. a cell that comprises anantigen, and a second part that can bind to a cell, e.g. a leukocyte,e.g. an APC. The molecule may have any of the characteristics taught inthe descriptions of methods and compositions herein. Preferably thefirst and second parts are heterologous to each other. The molecule maybe, e.g., a recombinant polypeptide expressed in a mammalian cell, aninsect cell, a plant cell, a yeast cell, or a bacterial cell.

[0045] The invention also encompasses a method of modulating an immuneresponse in an animal comprising the step of expressing in an animal,e.g. expressing in a host cell of the animal, a multifunctional moleculeof the invention, e.g. a polypeptide which comprises a first part thatcan bind to a antigen bearing target and a second part that can bind toa cell. According to the invention, “expressing in an animal” means“causing to be present in an animal”. When the molecule is apolypeptide, it is preferably expressed by introducing into the hostcell, in vivo or ex vivo, a nucleic acid encoding the polypeptide. Ifthe nucleic acid is introduced into the host cell ex vivo, the host cellmay subsequently be administered to the animal. In a preferredembodiment, the method further comprises administering to the animal theantigen to which the immune response is modulated. For example, theantigen may be administered to the animal as part of a composition whichfurther comprises a nucleic acid that encodes the multifunctionalmolecule. In another preferred embodiment, the antigen is alreadypresent in the animal at the time the multifunctional molecule isexpressed. In yet another preferred embodiment, the antigen isadministered to the animal after administration of the multifunctionalmolecule. In still other preferred embodiments, the antigen is expressedin the animal, e.g. by administering to the animal a compositioncomprising a nucleic acid encoding the antigen, either before or afterexpression of the multifunctional molecule in the animal. In anotherembodiment, nucleic acid sequences encoding the multifunctional moleculeand the antigen are introduced into one or more host cells of theanimal, e.g. by administering to the animal a composition comprisingthose nucleic acid sequences.

[0046] As used herein, the term “modulating an immune response” to aselected antigen using the methods and compositions of the inventionmeans rendering the response more or less efficient, more or less rapid,greater or lesser in magnitude, and/or more or less easily induced thanthe response obtained from administration of a composition which isidentical in every respect except that it does not comprise amultifunctional molecule of the invention. In a preferred embodiment,the response is between about 5 and 100%, or preferably between about 5and 50% or more preferably between about 5 and 25% more or lessefficient, more or less rapid, greater or lesser in magnitude, and/ormore or less easily induced than the response obtained fromadministration of a composition which is identical in every respectexcept that it does not comprise a multifunctional molecule of theinvention.

[0047] The term “modulate the immune response” may refer tostimulation/activation of an immune response to a selected antigen, orit may refer to suppression, elimination, or attenuation of an immuneresponse to a selected antigen. In a preferred embodiment, modulatingthe immune response results in stimulation/activation of an immuneresponse to a selected antigen by about at least 5%, or preferablybetween 5 and 50% or more preferably between 50 and 100%, as compared toan immune response in the absence of vaccination, or it may result insuppression, elimination, or attenuation of an immune response to aselected antigen by about at least 5%, or preferably between 5 and 50%or more preferably between 50 and 100%, as compared to an immuneresponse in the absence of vaccination. In some cases, one immuneresponse to an antigen (e.g. a Th1 response) may be increased whileanother immune response to the same antigen (e.g. a Th2 response) may bediminished.

[0048] The invention also encompasses a composition comprising amultifunctional molecule of the invention and antigen bearing target,e.g. a virus, a prion, or a cell. Preferably, when the antigen bearingtarget is a cell, the multifunctional molecule is exogenous to the cell.The multifunctional molecule may be heterologous to the cell. In oneembodiment, the multifunctional molecule is expressed within the cell,e.g. from a recombinant nucleic acid within the cell. The invention alsoencompasses a cell comprising a nucleic acid encoding a multifunctionalmolecule of the invention. The multifunctional molecule may have any ofthe characteristics set forth herein. Cells useful in the inventioninclude, for example, malignant cells, benign tumor cells, lymphocytes,e.g. B or T lymphocytes which may be pathogenic and/or autoreactive,cells expressing an antigen from an exogenously introduced nucleic acidmolecule, eukaryotic cells such as mammalian cells, human cells,fibroblasts, insect and fungal cells, and prokaryotic cells such asbacterial cells. Examples of viruses useful in the invention include,e.g., retroviruses such as human immunodeficiency viruses 1 and 2;herpesviruses such as herpes simplex viruses 1 and 2, cytomegalovirus,and varicella zoster virus; human papilloma virus; rabies virus;rotavirus; influenza viruses A, B, and C; hepatitis viruses A, B, C, andE or delta agent; adenoviruses; measles virus; mumps virus; polio virus;rubella virus; parainflunza viruses; coxsackie viruses A and B; variolavirus; yellow fever virus; dengue and other hemorrhagic fever viruses;West Nile fever virus; Eastern equine encephalitis virus; Western equineencephalitis virus; Venezuelan equine encephalitis virus; Japaneseencephalitis virus; rhinoviruses; and foot and mouth disease virus.Prions include the agents of scrapie, kuru, and bovine spongiformencephalitis. The cell, virus, or prion may be attenuated, i.e. renderednon-pathogenic, by, e.g,, killing, irradiation, chemical fixation,passaging in culture with selection for diminished pathogenicity, orgenetic manipulation. Preferably, the composition further comprises aleukocyte, e.g. a monocyte, a cell of monocytic lineage, a macrophage,or a dendritic cell or another APC.

[0049] Preferably, in the inventive methods and compositions, the cellis substantially unable to divide in vitro. “Substantially unable todivide in vitro” means that the cell divides at a rate that is less thanabout 50% of the rate of division of corresponding cells which are nottreated to prevent cell division. In a preferred embodiment, the celldivides at a rate that is less than about 30-50% of the rate of divisionof corresponding cells which are not treated to prevent cell division.

[0050] Preferably, the composition is substantially free of culturemedium. As used herein, “culture medium” refers to medium that is usedin cell culture containing at least 2% animal serum, such as fetal calfserum.

[0051] More particularly, the present invention provides amultifunctional molecule which is a fusion polypeptide comprising: alectin which comprises at least about 10 contiguous amino acids of aninfluenza virus hemagglutinin, and at least about 5 contiguous aminoacids of a naturally occurring GM-CSF molecule.

[0052] In one embodiment, the lectin is N-terminal to the contiguousamino acids of a naturally occurring GM-CSF molecule.

[0053] In an alternate embodiment, the lectin is C-terminal to thecontiguous amino acids of a naturally occurring GM-CSF molecule.

[0054] In one embodiment, the lectin comprises at least about 10contiguous amino acids of the HA1 domain of an influenza virushemagglutinin.

[0055] In one embodiment, the lectin is the HA1 domain of an influenzavirus hemagglutinin.

[0056] Preferably, the influenza virus hemagglutinin is a hemagglutininof an influenza A virus. In other preferred embodiments the influenzavirus hemagglutinin is a hemagglutinin of an influenza B or influenza Cvirus.

[0057] In one embodiment, the influenza virus hemagglutinin is of asubtype from a virus that infects humans. Preferably, the influenzavirus hemagglutinin is of an H1 subtype. Still more preferably, theinfluenza virus hemagglutinin is from the influenza A strain PR/8/34.

[0058] In one embodiment, the influenza virus hemagglutinin is of an H2subtype.

[0059] In one embodiment, the influenza virus hemagglutinin is of an H3subtype.

[0060] In one embodiment, the influenza virus hemagglutinin is of asubtype from a virus that does not infect humans.

[0061] In one embodiment the fusion polypeptide comprises the entireamino acid sequence of a naturally occurring GM-CSF molecule.

[0062] Preferably, the GM-CSF molecule is a murine GM-CSF. Still morepreferably, the GM-CSF molecule is a human GM-CSF.

[0063] The present invention encompasses a method of modulating animmune response in an animal comprising the step of administering acomposition comprising a multifunctional molecule, e.g. a polypeptide,e.g. a fusion polypeptide, which comprises a first part that can bind toa antigen bearing target and a second part that can bind to a cell. In apreferred embodiment, the composition further comprises an antigen, animmune response to which is modulated by administration of thecomposition. The antigen may be, for example, a polypeptide (e.g. arecombinant polypeptide), a lipid (e.g. a glycolipid), or a carbohydrate(e.g. a polysaccharide or a component of a bacterial or fungal cellwall). The composition therefore comprises an antigen bearing target,whether, e.g., a homogeneous antigen or a heterogeneous structure suchas a cell or a virus. When the antigen bearing target is a cell, it maybe autologous, syngeneic, allogeneic, or xenogeneic to the animal. Inother preferred embodiments, the antigen is already present in theanimal at the time the molecule is administered, and/or the antigen isadministered to the animal prior to administration of the molecule. Inyet another preferred embodiment, the antigen is administered to theanimal after administration of the molecule.

[0064] Preferably, the composition comprises multifunctional moleculeswhich are not bound to an antigen bearing target. In a preferredembodiment, the composition further comprises an antigen bearing target,e.g. a cell. In one embodiment of the invention, the compositioncomprises multifunctional molecules, some of which are bound to aantigen bearing target, e.g. to the surface of a cell, and some of whichare external to and not bound to any target. In another embodiment, thecomposition comprises a multifunctional molecule and further comprises aportion of a cell, e.g. a membrane fraction of a cell (i.e., an antigenbearing target). In yet another embodiment, the composition comprises amultifunctional molecule and further comprises a multiplicity ofdifferent molecules derived from a cell, as is found, e.g., in a celllysate. Cells may be lysed, for example, by freezing and thawing,preferably repeatedly. In a preferred embodiment, the composition iscell-free.

[0065] The present invention further encompasses a method of vaccinatinga mammal to a selected antigen comprising administering to the animal avaccine composition comprising a multifunctional molecule of theinvention comprising a first part which is a lectin, and a second partwhich is a ligand for a cell surface receptor, e.g. a cell surfacereceptor of an APC. Preferably, the lectin can bind to an antigenbearing target which comprises the antigen.

[0066] In one embodiment, the invention provides a method of vaccinatinga mammal to a selected antigen comprising removing at least one cellfrom the mammal, wherein the cell comprises the antigen, contacting thecell ex vivo with a multifunctional molecule comprising a first partwhich is a lectin and is capable of binding to at lease one carbohydratemolecule on the surface of the antigen bearing cell, and a second partwhich is a ligand for a cell surface receptor of an APC, so as to forman antigen bearing cell/multifunctional molecule complex; and placingthe complex back into the mammal.

[0067] In a preferred embodiment, the composition comprises an antigen,an immune response to which is modulated by administration of thecomposition.

[0068] The present invention provides a method for juxtaposing an APCwith an antigen bearing target comprising: contacting an APC and antigenbearing target with a multifunctional molecule comprising a first partcomprising a lectin which is able to bind to at least one carbohydratemoiety on the antigen bearing target and a second part comprising aligand for a cell surface receptor on the APC. Preferably, themultifunctional molecule is first contacted with the antigen bearingtarget and the resulting antigen bearing target/multifunctional moleculecomplex is subsequently contacted with the APC. In one embodiment theantigen bearing target is a cell from an animal comprising an antigen,and is contacted with the multifunctional molecule ex vivo underconditions which permit the binding of the lectin to at least onecarbohydrate moiety of the cell. The resulting multifunctionalmolecule/antigen bearing cell complex is then administered back to theanimal from which the antigen bearing cell was derived wherein it isable to bind to a cell surface receptor on an APC via the ligand portionof the multifunctional molecule, thereby juxtaposing the antigen bearingtarget and the APC.

[0069] “Juxtaposition”, in the context of the present invention,includes but is not limited to physical contact. An APC and antigenbearing target are “juxtaposed” with one another if they aresufficiently close for the APC to internalize the antigen bearingtarget. An APC and antigen bearing target are also “juxtaposed” if theyare separated by no more that 20 μm, preferably no more than 10 μm, andstill more preferably no more than 5 μm, and more preferably no morethan 1 μm.

[0070] As used herein, “contacting” refers to admixing in vitro or invivo.

[0071] The invention also encompasses a method of modulating an immuneresponse to an antigen comprising contacting in vitro an antigen bearingtarget, a multifunctional molecule of the invention, and an APC andadministering the resultant composition to a subject. In one embodimentthe antigen bearing target/multifunctional molecule complex is contactedwith an APC for a time sufficient to permit internalization of theantigen bearing target by the APC. In other embodiments the antigenbearing target/multifunctional molecule complex is contacted with an APCfor a time that allows internalization of less than about 80%, less thanabout 60%, less than about 40%, less than about 20%, less than about10%, or less than about 5% of the antigen bearing target by the APC.Methods for determining the amount of target internalized, e.g. bymeasuring the amount remaining outside the APC and subtracting from thestarting amount, are well-known in the art. Preferably, the antigenbearing target/multifunctional molecule complex is contacted with an APCfor less than about 10 minutes, less than about 30 minutes, less thanabout 60 minutes, less than about 90 minutes, less than about 120minutes, or less than about 180 minutes.

[0072] As used herein, “time sufficient to permit internalization”refers to a period of time that is of a sufficient duration to allowinternalization of the selected antigen or antigen bearing targtet bythe APC (for example, no more than about fourteen days, or seven days,or five or three days, or as little as about 24, 12, 6, 3, 2 or 1 hour,or even as little as about 30, 20, 10, 5, or 1 minute).

[0073] The invention also encompasses a method of attaching a ligand fora cell surface polypeptide to an antigen bearing target comprisingadmixing the antigen bearing target with a multifunctional moleculewhich comprises the ligand. The invention also encompasses a method ofattaching an amino acid sequence to an antigen bearing target comprisingadmixing the antigen bearing target with a fusion polypeptide whichcomprises the amino acid sequence and further comprises a lectin. Theinvention also encompasses a composition comprising an antigen bearingtarget admixed with a fusion polypeptide which comprises a first aminoacid sequence which is not a lectin and a second amino acid sequencewhich comprises a lectin.

[0074] The invention also comprises methods of producing amultifunctional molecule of the invention in each of the following celltypes: a yeast cell, a mammalian cell, a bacterial cell, an insect cell.Each of these methods comprises the step of introducing a nucleic acidencoding a multifunctional molecule into the respective cell type, astaught hereinbelow.

[0075] The invention also encompasses methods of detecting orquantifying a multifunctional molecule of the invention comprisingcontacting the multifunctional molecule with an antibody or other ligandthat binds to the multifunctional molecule. Such methods include ELISAassays and flow cytometry, as described hereinbelow. Preferably, themultifunctional molecule to be detected or quantitated is bound to anantigen bearing target.

DETAILED DESCRIPTION

[0076] The present invention is based, in part, on the discovery that amultifunctional fusion protein comprising a first polypeptide which is alectin and a second polypeptide which is a ligand of a cell surfacereceptor of an APC, can effectively target an antigen bearing target,such as a cell bearing an antigen of interest, to an APC, wherein theantigen is engulfed by the APC, and an appropriate immune response tothe antigen is mounted by an animal to which the multifunctionalmolecule is administered.

[0077] Accordingly, the present invention provides a method forvaccinating a mammal comprising administering to the animal a vaccinecomposition comprising a multifunctional molecule of the inventioncomprising a first part which is a lectin and which can bind to a targetbearing the antigen, and a second part which is a ligand for a cellsurface receptor of an APC. In one embodiment, the method comprisesremoving at least one cell from the mammal, wherein the cell comprisesthe antigen, contacting the cell ex vivo with a multifunctional moleculecomprising a first part which is a lectin and is capable of binding toat lease one carbohydrate molecule on the surface of the antigen bearingcell, and a second part which is a ligand for a cell surface receptor ofan APC, so as to form an antigen bearing cell/multifunctional moleculecomplex; and placing the complex back into the mammal.

[0078] Multifunctional Molecules

[0079] The present invention encompasses a multifunctional moleculecomprising a first part which can bind to an antigen bearing target, anda second part which is a ligand for a cell surface protein of a cell,e.g. an antigen presenting cell. Preferably, the first part which canbind to an antigen bearing target is a lectin which binds to at leastone carbohydrate molecule present on the antigen bearing target.Preferably the lectin is an influenza hemaggluti nin and binds to sialicacid residues present on the antigen bearing target. Preferably, theligand of a cell surface protein of an antigen presenting cell isselected from an opsonin, a cytokine, a ligand for a CD40 molecule, anadhesion molecule, a defensin, a heat shock protein, or acounterreceptor for a T cell costimulatory molecule. Cell surfacemolecules which can act as receptors for the second part of themultifunctional molecule include CD40 molecules and specific receptorsfor an opsonin, a cytokine, an adhesion molecule, a defensin, a heatshock protein, or a counterreceptor for a T cell costimulatory molecule,and also include, but are not limited to the cell surface moleculeslisted in Apendix I and II.

[0080] Lectins

[0081] The multifunctional molecule which comprises first and secondparts can comprise a first part which comprises a lectin and a secondpart that can bind to a leukocyte, e.g. an APC, e.g. a cell of monocyticlineage or a dendritic cell (which may itself be of monocytic lineage).A “lectin”, according to the invention, is a molecule or part of amolecule, e.g. an amino acid sequence, which can bind to a carbohydrate,e.g. a polysaccharide. Families of naturally occurring lectins include:

[0082] 1) Galectins, a rapidly growing family of animal lectins. All ofthem share galactose-specificity.

[0083] 2) Calcium-dependent (C-type) animal lectins, an extremely largefamily composed of members having diverse structures and functions.

[0084] 3) Among this C-type lectin family, selectins form adistinguishable subfamily by their specific function in leukocyteadhesion to endothelial cells through sialyl-LewisX recognition.

[0085] 4) Collectins, another subfamily of C-type lectins specific formannose, which have a unique structure consisting of a C-type lectindomain and a collagen-like domain. They are involved in innate immunity.

[0086] 5) Invertebrates are known to contain various lectins in theirbody fluids, probably as body-protection factors. Recently, some lectinsfrom an echinoderm were found to show hemolytic activity.

[0087] 6) Annexins, a group of proteins having affinity to lipids thatwere recently shown to be lectins showing binding to glycosaminoglycans.

[0088] 7) The legume lectin family, which consists of a large number ofmembers, such as ConA, with variable saccharide specificity comparableto C-type lectins.

[0089] 8) Ricin, the first lectin investigated in Russia more than 100years ago. It is now evident that the ricin family has many otherhomologous members which differ in either toxicity or sugar-bindingspecificities.

[0090] Thus, a multifunctional molecule of the invention may bind to oneor more carbohydrates. Carbohydrates to which lectins may bind alsoinclude, for example, carbohydrates comprising lactose, D-mannose,D-glucose, D-fucose, L-fucose (e.g. alpha-L-fucose), D-galactose, bloodgroup A oligosaccharides, blood group B oligosaccharides, saccharidescomprisingalpha-D-Gal(1->3)[alpha-Lfuc(1->2)]-beta-D-Gal(1->¾-beta-D-GlcNAc,saccharides comprising alpha-sialyl-[2->3]-lactose, alpha-D-mannosylglycoconjugates, alpha-NeuNAc-[2->6]-Gal, alpha-NeuNAc-[2->6]-GalNAc,alpha-NeuNAc-[2->3]-Gal, N-acetyl-beta-D-glucosamine, terminalalpha-D-galactosyl residues, terminal beta-D-galactosyl residues,N-acetyllactosamine, terminal alpha-D-mannosyl residues,N-acetyl-beta-D-glucosamine, terminal N-acetyl-D-galactosamine,N-acetylneuraminic acid, and terminal alpha-D-galactosaminyl residues.

[0091] The multifunctional molecule which comprises a lectin maycomprise, for example, the whole of a naturally occurring lectin or aportion of a naturally occurring lectin, e.g about (or at least about)5, 8, 10, 12, 15, 20, 25, 35, 50, 60, 70, 80, 100, or 120 contiguousamino acids of a naturally occurring polypeptide lectin. In oneembodiment the multifunctional molecule comprises a carbohydrate-bindingdomain of a naturally occurring lectin, i.e., a portion of a lectin thatcan bind to a carbohydrate in the absence of the remainder of thelectin. In another embodiment the lectin may be non-naturally occurring,e.g. identified from an artificial library of molecules or designed bymodifying the structure of a naturally occurring lectin.

[0092] Lectins known as “hemagglutinins” bind to carbohydrates onerythrocytes, e.g. blood group antigens, and when incubated with thesecells cause them to aggregate. The influenza virus hemagglutinin, forexample, binds to sialic acid. There are at least 15 known influenzahemagglutinin subtypes, defined by their distinct antigenic properties.Any of these subtypes, designated, e.g., H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, and H15, may provide amino acid sequencesuseful in the compositions and methods of the invention. In oneembodiment of the invention, the hemagglutinin is of a subtype from avirus that infects humans, e.g. H1, H2, or H3. In another embodiment,the hemagglutinin is of a subtype from a virus that does not infecthumans, e.g. one of H4 through H15. Amino acid sequences can vary up toabout 20% for influenza hemagglutinins within a given subtype, and canvary between about 30% and about 70% for influenza hemagglutinins fromdifferent subtypes. Methods for determining amino acid sequence homologyare known to those of skill in the art. Examples of other software thatcan perform sequence comparisons to determine the % identity betweenhemagglutanin variants (or variants of any portion of themultifunctional molecules disclosed herein) include, but are not limitedto, the BLAST package (Ausubel et al., 1995, Short Protocols inMolecular Biology, 3rd Edition, John Wiley & Sons), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching.

[0093] Although the final % homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied. It is preferred to use the public default values for the GCGpackage, or in the case of other software, the default matrix, such asBLOSUM62.

[0094] Advantageously, the BLAST algorithm is employed, with parametersset to default values. The BLAST algorithm is described in detail inAltschul et al., (1990) J. Mol. Biol. 215:403-410, which is incorporatedherein by reference. The search parameters are defined as follows, andcan be advantageously set to the defined default parameters.

[0095] Advantageously, “substantial identity” when assessed by BLASTequates to sequences which match with an EXPECT value of at least about7, preferably at least about 9 and most preferably 10 or more. Thedefault threshold for EXPECT in BLAST searching is usually 10.

[0096] BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (Karlin and Altschul 1990,Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. USA 90:5873-7) with a few enhancements. The BLASTprograms are tailored for sequence similarity searching, for example toidentify homologues to a query sequence. For a discussion of basicissues in similarity searching of sequence databases, see Altschul et al(1994) Nature Genetics 6:119-129.

[0097] The five BLAST programs available through the National Institutesof Health (NIH; Bethesda, Md.) perform the following tasks: blastpcompares an amino acid query sequence against a protein sequencedatabase; blastn compares a nucleotide query sequence against anucleotide sequence database; blastx compares the six-frame conceptualtranslation products of a nucleotide query sequence (both strands)against a protein sequence database; tblastn compares a protein querysequence against a nucleotide sequence database dynamically translatedin all six reading frames (both strands); tblastx compares the six-frametranslations of a nucleotide query sequence against the six-frametranslations of a nucleotide sequence database.

[0098] BLAST uses the following search parameters:

[0099] HISTOGRAM—Display a histogram of scores for each search; defaultis yes. (See parameter H in the BLAST Manual).

[0100] DESCRIPTIONS—Restricts the number of short descriptions ofmatching sequences reported to the number specified; default limit is100 descriptions. (See parameter V in the manual page).

[0101] EXPECT—The statistical significance threshold for reportingmatches against database sequences; the default value is 10, such that10 matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

[0102] CUTOFF—Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

[0103] ALIGNMENTS—Restricts database sequences to the number specifiedfor which high-scoring segment pairs (HSPs) are reported; the defaultlimit is 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

[0104] MATRIX—Specify an alternate scoring matrix for BLASTP, BLASTX,TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff &Henikoff, 1992). The valid alternative choices include: PAM40, PAM120,PAM250 and IDENTITY. No alternate scoring matrices are available forBLASTN; specifying the MATRIX directive in BLASTN requests returns anerror response.

[0105] STRAND—Restrict a TBLASTN search to just the top or bottom strandof the database sequences; or restrict a BLASTN, BLASTX or TBLASTXsearch to just reading frames on the top or bottom strand of the querysequence.

[0106] FILTER—Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Claverie & States (1993) Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(NIH). Filtering can eliminate statistically significant butbiologically uninteresting reports from the blast output (e.g., hitsagainst common acidic-, basic- or proline-rich regions), leaving themore biologically interesting regions of the query sequence availablefor specific matching against database sequences.

[0107] Low complexity sequence found by a filter program is substitutedusing the letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) andthe letter “X” in protein sequences (e.g., “XXXXXXXXX”).

[0108] Filtering is only applied to the query sequence (or itstranslation products), not to database sequences. Default filtering isDUST for BLASTN, SEG for other programs.

[0109] It is not unusual for nothing at all to be masked by SEG, XNU, orboth, when applied to sequences in SWISS-PROT, so filtering should notbe expected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

[0110] NCBI-gi—Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

[0111] Most preferably, sequence comparisons are conducted using thesimple BLAST search algorithm provided by the NIH. In some embodimentsof the present invention, no gap penalties are used when determiningsequence identity.

[0112] Influenza hemagglutinin is expressed as a single polypeptidechain, designated HA0, which trimerizes post-translationally. HA0 isproteolytically cleaved to yield two domains, HA1 and HA2, which aredisulfide-bonded to each other. HA1 comprises significant sialic acidbinding activity, while HA2 is anchored to the viral membrane andfacilitates fusion of this membrane with a host cell membrane. Inpreferred embodiments of the invention, the multifunctional moleculecomprising first and second parts comprises an amino acid sequence of anHA1 domain.

[0113] Additional examples of lectin molecules useful in the presentinvention include, but are not limited to, those lectins shown in Table1, and variants thereof having at least 50%, 70%, 90%, and up to 99%sequence homology with the sequences of the lectins shown in Table 1.TABLE 1 KEY NAME ABBREVIATION CLASS LECTIN CODE [1] /—/— QuailIntestinal — LECa.Ggg.Sss.xx.Xxxx. Lectin. [2] /—/— Porcine Heart Lectin— LECa.Ggg.Sss.xx.Xxxx. (PHL). [3] /—/— Hepatic beta- S-lectin orGLTa.Ggg.Sss.xx.Xxxx. galactoside binding Galectin. lectins. [4] /—/—Mammalian Brain S-lectin or GLTa.Ggg.Sss.xx.Xxxx. Beta-Galactoside-Galectin. binding Lectin. [5] Aaptos papillata. — —LECi.Ada.Pap.xx.Xxxx. [6] Abelmoschus — — LECp.Abe.Esc.xx.Xxxx.esculentus. [7] Abramis brana. — — LECp.Abr.Bra.xx.Xxxx. [8] Abrusprecatorius. APA; APA-A; APA- beta-trefoil lectin LECp.AbrPre.se.Cga1 C;Abrin. (APA); Type 2 (abrin) RIP. LECp.AbrPre.se.Cga2 (APA). [10]Achatina fulica. Achatina fulica Cold — LECi.Ach.ful.xx.Xsi1.Aggltutinin; achatinin-H. [13] Actinomyces — — LECf.Act.Vis.xx.Xga1.viscosus. [14] Adenia digitata. Modeccin. Type 2 RIP.LECp.AdeDig.ro.Cga1. [15] Adenia volksensii. Volkensin. Type 2 RIP.LECp.AdeVol.ro.Cga1. [16] Aegilops — Hevein domain LECp.Aeg.Gen.se.Hch1.geniculata. lectin, chitin binding. [19] Aegopodium APA. —LECp.Aeg.Pod.rh.Hga1. podagraria. [20] Aeromonas — —LECb.Aer.Sal.xx.Xxxx. salmonicida. [21] Afzelia africana. — —LECz.Afz.Afr.xx.Xxxx. [22] Agardhiella tenera. — — LECz.Aga.Ten.xx.Xxxx.[23] Agaricales. — — LECz.Aga.sss.xx.Xxxx. [24] Agaricus bisporus.ABA-I, ABA-II, — LECf.Aga.Bis.xx.Xga1. ABA-III, ABA-IV. [25] Agaricusblazei. — — LECf.Aga.Bla.xx.Xxxx. [26] Agaricus — —LECf.Aga.Cam.xx.Xxxx. campestris. [27] Agaricus edulis. — —LECf.Aga.Edu.xx.Xxxx. [28] Agrobacterium — — LECu.Agr.Rad.xx.Xxxx.radiobacter. [29] Agrocybe aegerita. — — LECf.Agr.Aeg.xx.Xxxx. [30]Agropyrum AREL, ARLL. Hololectin; LECp.Agr.Rep.se.Hch1 repens. Monocot(AREL) mannose-binding LECp.Agr.Rep.le.Hch1 lectins. (ARLL). [31]Aleuria aurantia. — — LECf.Ale.Aur.xx.Xfu1. [32] Allium AAA. MonocotLECp.All.Asc.bu.Hma1. ascalonicum. mannose-binding lectins. [36] Alliumcepa. ACA. Monocot LECp.All.Cep.bu.Hma1. mannose-binding lectins. [37]Allium moly. AMA. Monocot LECp.All.Mol.bu.Hma1. mannose-binding lectins.[38] Allium porrum. APA. Monocot LECp.All.Por.le.Hma1. mannose-bindinglectins. [39] Allium sativam. ASA. Monocot LECp.All.Sat.bu.Hma1Mannose-binding (ASA-I) lectin. LECp.All.Sat.bu.Hma1 (ASA-I)LECp.All.Sat.bu.Hma1 (ASA-I) LECp.All.Sat.bu.Hma1 (ASA-I)LECp.All.Sat.bu.Hma2 (ASA-II) LECp.All.Sat.le.Hma1 (ASA-III)LECp.All.Sat.ro.Hma1 (ASA-IV). [40] Allium ursinum. AUA-I, AUA-II,Monocot LECp.All.Urs.bu.Hma1 AUA-III, AUA-Ir, Mannose-binding (AUA-I)AUA-L, AUA-Iir. lectin LECp.All.Urs.bu.Hma2 (AUA-II)LECp.All.Urs.le.Hma1 (AUA-L) LECp.All.Urs.ro.Hma1 (AUA-Ir)LECp.All.Urs.ro.Hma2 (AUA-IIr). [42] Allium vineale. AVA. MonocotLECp.All.Vin.bu.Hma1. Mannose-binding lectin. [43] Allomyrina — —LECi.All.Dic.xx.Xxxx. dichotoma. [44] Alocasia indica. — —LECp.Alo.Ind.tu.Hcu1. [45] Aloe arborescens. Aloctin, AAA. AAA: MonocotLECp.Alo.Arb.le.? Mannose-binding (Aloctin-A) proteins Aloctin-LECp.Alo.Arb.le.Hma1 A: u. (AAA). [46] Amaranthus ACA, Amaranthin,beta-trefoil lectin, LECp.Ama.Cau.se.Hga1. caudatus. ACL. Amaranthingroup. [47] Amaranthus — Amaranthin LECp.Ama.Cru.se.Hga1. cruentus.group. [48] Amaranthus AHML, Amaranthin. AmaranthinLECp.Ama.Hyp.xx.Xgal1. hypochondriacus. group. [49] Amaranthus —Amaranthin LECp.Ama.Leu.se.Hga1. leucocarpus. group. [50] AmaranthusASL. Amaranthin LECp.Ama.Spi.se.Hga1. spinosus. group. [51] AmphicarpaeaABrA. Legume lectins. LECp.Amp.Bra.se.Hma1. bracteata. [52] Anadaragranosa. Anadarin MS. — LECi.Ana.Gra.xx.Xsi1. [53] Anguilla anguilla.AAL. — LECi.Ang.Ang.xx.Xfu1. [54] Anthocidaris — Novel, uniqueLECi.Ant.Cra.xx.Xxxx. crassispina. lectin class. [55] Anthocidaris — —LECi.Ant.Cra.xx.Xxxx. crassispina Ovum. [57] Apium graveolens. — —LECp.Api.Gra.xx.Xxxx. [58] Aplysia — — LECi.Apl.Dac.xx.Xxxx.dactylomela. [59] Aplysia depilans. — — LECi.Apl.Dep.xx.Xga1. [60]Aplysina archeria. — — LECu.Apl.Arc.xx.Xxxx. [61] Arachis hypogea. PNA,GNL, MNL, All Arachnis LECp.Ara.Hyp.se.Hga1 PRA-I, PRA-II. lectins areclassed (PNA) as legume lectins LECp.Ara.Hyp.no.Hga1 (GNL)LECp.Ara.Hyp.se.Hga1 (MNL) LECp.Ara.Hyp.se.Hga1 (PRA-I)LECp.Ara.Hyp.se.Hga1 (PRA-II). [62] Araucaria Lectin I, Lectin II. —LECp.Ara.Bra.se.Hmg1 brasiliensis. (Lectin I) LECp.Ara.Bra.se.Hmg2(Lectin II). [63] Arion — — LECi.Ari.Emp.xx.Xxxx. empiricorum. [64]Arisaema ACA. — LECp.Ari.Con.tu.Hcu1. consanguineum. [65] Arisaema ACmA.— LECz.Ari.Cur.tu.Hcu1. curvatum. [66] Arthrobotrys AOL. —LECf.Art.Oli.xx.Xxxx. oligospora. [68] Artocarpus hirsuta. — —LECp.Art.Hir.xx.Xxxx. [69] Artocarpus incisa. — — LECp.Art.Inc.xx.Xxxx.[70] Artocarpus Jacalin, AIA, KM+, beta-prism plantLECp.Art.Int.se.Hga1. integrifolia. Artocarpin. lectin, Jacalin- relatedlectins. [71] Artocarpus Artocarpin, ALA-I, Jacalin-relatedLECp.Art.Lak.se.Hga1. lakoocha. ALA-II. lectins. [72] Arum maculatum.AMA. Monocot binding LECp.Aru.Mac.tu.Hma1. lectins. [73] Ascaris — —LECi.Asc.Lum.xx.Xxxx. lumbricoides. [74] Asparagus — —LECp.Asp.Off.xx.Xxxx. officinalis. [75] Bacillus — —LECb.Bac.Pols.xx.Xxxx. polymyxa. [76] Bacterioides — —LECb.Bac.Fra.xx.Xxxx. fragilis. [77] Bandeiraca BS-I, BS-I-A4, BS-I- —LECp.Ban.Sim.xx.Xxxx. simplicifolia. B4, BS-II. [78] Basidiomycotina. —— LECf.Bas.Sss.xx.Xxxx. [79] Bauhinia purpurea. BPA. Legume lectin.LECp.Bau.Pur.se.Hga1. [80] Bauhinia — — LECz.Bau.Tom.xx.Xxxx. tomentosa.[81] Beauveria — — LECf.Bea.Bas.xx.Xsi1. bassiana. [82] Beta vulgaris. —— LECp.Bet.Vul.xx.Xxxx. [83] Beta vulgaris. — — LECp.Bet.Vul.xx.Xxxx.[84] Biomphalaria BGL-I, BGL-II. — LECp.Bio.Gla.xx.Xxxx. glabrata. [85]Biomphalaria — — LECi.Bio.Gla.xx.Xxxx. glabrata. [86] Birgus latro. — —LECz.Bir.Lat.xx.Xxxx. [87] Blaberus BDL1, BDL2, BDL3. —LECi.Bla.Dis.xx.Xxxx. discoidalis. [88] Bordetella Pertussis toxin 1PRT.— LECz.Ggg.Sss.xx.Xxxx. pertussis. [89] Bos Taurus. Mannose 6-phosphateP-lectin. LECa.Bos.Tau.xx.Xxxx. receptor (1C39). [90] Bos taurus. BovineConglutinin. C-lectin or LECa.Bos.Tau.xx.Xxxx. Collectin. [91] Bostaurus. Bovine collectin-43 C-lectin or Leca.Bos.Tau.xx.Xxxx. (CL-43).Collectin. [92] Botryllus — S-lectin. LECi.Bot.Sch.xx.Xxxx. schlosseri.[93] Botrytis cinerea. — — LECz.Bot.Cin.xx.Xxxx. [94] Bowringia BMA.Legume lectins. LECp.Bow.Mil.se.Hmg1. milbraedii. [95] BrachypodiumBsyL. Chitin-binding LECp.Bra.Syl.se.Hch1. sylvaticum. lectins. [96]Bradyrhizobium — — LECp.Bra.Jap.xx.Xga1. japonicum. [97] Branchiostoma —— LECi.Bra.Lan.xx.Xxxx. lanceolatum. [98] Brassica — —LECp.Bra.Cam.xx.Xxxx. campetsris. [99] Brassica — —LECp.Bra.Nap.xx.Xxxx. napobrassica. [100] Brassica napus. — —LECp.Bra.Nap.xx.Xxxx. [101] Bryonia dioica. BDA. — LECp.Bry.Dio.tu.Hga1.[113] Cancer — — LECi.Can.Ant.xx.Xsi1. antennarius. [114] Candidaalbican Adhesins. — LECf.Can.Alb.xx.Xfu1. adhesin. [115] Cannageneralis. — — LECp.Can.Gen.rh.Hma1. [116] Capnocytophaga — —LECu.Cap.Gin.xx.Xxxx. gingivalis Actinomyces israelii Coaggregationagglutinin. [117] Capsicum annum. — — LECp.Cap.Ann.xx.Xxxx. [118]Caragana CAA-I, CAA-II. — LECp.Car.Arb.se.Hga1 arborescens. (CAA-I)LECp.Car.Arb.se.Hga2 (CAA-II). [119] charhinus — — LECa.Car.Spr.xx.Xxxx.ingeri. [120] Carcinoscorpious L10; carcinoscorpin. —LECi.Car.Rot.xx.Xsi1. rotundacauda. [121] Carica papya. — —LECp.Car.Pap.xx.Xxxx. [123] Carum carvia. — — LECp.Car.Car.xx.Xxxx.[124] Carybdea alata — — LECi.Car.Ala.xx.Xxxx. Hemolysin. [125] Castaneacrenata. CCA. — LECp.Cas.Cre.xx.Xxxx. [126] Cepaea hortensis CHA-I. —LECi.Cep.Hor.xx.Xxxx. [127] Channa punctatus. — — LECa.Cha.Pun.xx.Xxxx.[129] Chelidonium — — LECp.Che.Maj.se.Hch1. majus. [132] Chicorium — —LECp.Chi.Int.xx.Xxxx. intybus. [133] Cholla opuntia. — —LECp.Cho.Opu.xx.Xxxx. [134] Cicer arietinum. CAA. —LECp.Cic.Ari.se.Hcu1. [135] Cinachyrella — — LECi.Cin.All.xx.Xxxx.alloclada. [136] Cinnamonum — — LECp.Cin.Cam.xx.Xxxx. camphora. [137]Citrullus vulgaris. — — LECp.Cit.Vul.xx.Xxxx. [139] Citrus aurantium. —— LECp.Cit.Aur.se.Cnd1. [140] Citrus aurantium. — —LECp.Cit.Aur.xx.Xxxx. [141] Citrus medica. — — LECp.Cit.Med.xx.Xxxx.[142] Cladrastis lutea. CLA-I, CLA-II. — LECp.Cla.Lut.ba.Hmg1 (CLA-I)LECp.Cla.Lut.ba.Hmg2 (CLA-II). [143] Clerodendron CTA. —LECp.Cle.Tri.fr.Hga1. trichotomum. [144] Clitocyba — —LECf.Cli.Neb.xx.Xxxx. nebularis. [145] Clivia miniata. CMA. —LECp.Cli.Min.le.Hma1. [146] Clostridium — — LECb.Clo.Bot.xx.Xxxx.botulinum. [147] Clostridium tetani. Tetanus toxin —LECb.Ggg.Sss.xx.Xxxx. (1A8D). [148] Clupea harengus. — —LECa.Clu.Har.xx.Xxxx. [149] Coccinia grandis. CIA. —LECp.Coc.Gra.fr.Hch1. [151] Cocus nucifera. — — LECp.Coc.Nuc.xx.Xxxx.[152] Codium fragilis. — — LECu.Cod.Fra.xx.Xxxx. [153] Cofea arabica. —— LECp.Cof.Ara.xx.Xxxx. [154] Colchicum CAA. — LECp.Col.Aut.bu.Hcu1.autumnale. [155] Collybia velutipes. — — LECf.Col.Vels.xx.Xxxx. [156]Colocasia CEA. — LECp.Col.Esc.tu.Hma1. esculentum. [157] Congermyriaster. Congerin I, Congerin S-lectin. LECi.Con.Myr.xx.Xga1. II.[159] Conidiobolus — — LECf.Con.Obs.xx.Xga1. obscurus. [160] Coprinuscinereus. Cg1, Cg2. Galectin. LECf.Cop.Cin.xx.Xxxx. [161] Corbicula — —LECi.Cor.Flu.xx.Xxxx. fluminea Hemolysin. [163] Corylus avellania. — —LECp.Cor.Ave.xx.Xxxx. [164] Cratylia mollis. — — LECz.Cra.Mol.xx.Xxxx.[165] Crenomytilus CGL. — LECi.Cre.Gra.xx.Xxxx. grayanus. [166] Crocussativum. — — LECp.Cro.Sat.bu.Hma1. [167] Crocus vernus. CVA. —LECp.Cro.Ver.xx.Xxxx. [169] Crotalaria striata. — —LECp.Cro.Str.se.Hga1. [170] Crotolaria — — LECz.Cro.Aeg.xx.Xxxx.aegyptica. [171] Crotolaria falcata. — — LECz.Cro.Fal.xx.Xxxx. [172]Crotolaria juncea. — — LECp.Cro.Jun.se.Hga1. [174] Croton tiglium. — —LECp.Cro.Tig.se.Hcu1. [175] Cucumaria CEL-III. — LECi.Cuc.Ech.xx.Xxxx.echinata. [176] Cucumis — — LECp.Cuc.Cat.xx.Xxxx. catalupensis. [177]Cucumis melo. — — LECp.Cuc.Mel.xx.Xch1. [178] Cucumis sativus. — —LECp.Cuc.Sat.xx.Xch1. [180] Cucurbita ficifolia. — —LECp.Cuc.Fic.xx.Xxxx. [181] Cucurbita maxima. CMA, PP2. —LECp.Cuc.Max.ps.Hch1. [182] Cucurbita pepe. — — LECp.Cuc.Pep.xx.Xxxx.[183] Cucurbita pepo. CPA. — LECp.Cuc.Pep.fr.Hch1. [184] Cucurbitasativus. — — LECp.Cuc.Sat.xx.Xxxx. [185] Cydonia oblonja. — —LECp.Cyd.Obl.xx.Xxxx. [186] Cymbidium — — LECz.Cym.Hyb.le.Hma1. hybrid.[187] Cyphomandra — — LECp.Cyp.Bet.xx.Xxxx. betacea. [188] CytisisCMA-I, CMA-II. — LECp.Cyt.Mul.se.Hch1 multiflorus. (CMA-I)LECp.Cyt.Mul.se.Hfu1 (CMA-II). [189] Cytisus scoparius. CSA-I, CSA-II, —LECp.Cyt.Sco.se.Hga1 CMH-I, CMH-II. (CS-I) LECp.Cyt.Sco.se.Hga2 (CS-II).[190] Cytisus — — LECp.Cyt.Ses.se.Hch1 sessilfolius. (CSA-I)LECp.Cyt.Ses.se.Hga1 (CSA-II). [191] Dacrymycetales. — —LECz.Dac.sss.xx.Xxxx. [192] Dalbergia. — — LECz.Dal.sss.xx.Xxxx. [193]Datura innoxia. — — LECp.Dat.Inn.xx.Xxxx. [194] Datura DSA.Chitin-bindng LECp.Dat.Str.se.Hch1. stramonium. lectins. [195] Daucuscarrota. — — LECp.Dau.Car.xx.Xxxx. [196] Dendroaspis JML, Jameson's —LECi.Den.Jam.xx.Xga1. jamesoni. Mamba Venon. [198] Deuteromycetes. — —LECz.Deu.sss.xx.Xxxx. [199] Dicolea lehmani. — — LECz.Dio.Leh.xx.Xxxx.[200] Dictyostelium Discoidin I. — LECu.Dic.Dis.xx.Xxxx. discoideum.[201] Dictyostelium Purpurin. — LECu.Dic.Pur.xx.Xxxx. purpureum. [202]Didemnum DTL, DCL-I, DCL- — LECi.Did.Sss.xx.Xga1. candidum. II. [203]Dieffenbachia — — LECp.Dif.Seq.xx.Xxxx. sequina. [204] Dioclea — Legumelectin. LECp.Dio.Gra.xx.Xxxx. grandifolia. [205] Dioclea DLL-I, DLL-II,Legume lectin. LECp.Dio.Gui.xx.Xmg1. guianensis. DLL-III. [206] Diocleavirgata. — Legume lectin. LECz.Dio.Vir.xx.Xxxx. [207] Dolichos biflorus.DBA-S, DBA-R, Legume lectin. LECp.Dol.Bif.se.Hga1 DB-58, DB-57, (DBA)DB46. LECp.Dol.Bif.pl.Hcu1 (DB58) LECp.Dol.Bif.pl.Hcu2 (DB57)LECp.Dol.Bif.ro.?ga1 (DB46). [208] Drosophila. — — LECi.Dro.Meg.xx.Xxxx.[209] Dumasia. — — LECz.Dum.sss.xx.Xxxx. [210] Echinocereus — —LECp.Echi.Eng.xx.Xxxx. engelmanii. [211] Echis EMS16. —LECi.Ech.Mul.xx.Xxxx. multisquamatus. [212] Electrophorus Electrolectin.— LECi.Ele.Ele.xx.Xxxx. electricus. [213] Elymus — Hevein domainLECp.Ely.Can.se.Hch1. canadensis. lectin, chitin binding. [223]Erythrina velutina. — — LECp.Ery.Vel.xx.Xxxx. [224] Escherichia coli.Pili mannose-specific Verotoxin-1: LECb.Ech.Col.xx.Xxxx. FimH adhesinADP-ribosylating (1QUN),. toxins. [225] Euhadra — —LECz.Euh.Cal.xx.Xxxx. callizoma. [226] Euphorbia — —LECp.Eup.Sss.xx.Xxxx. characias. [227] Euphorbia — —LECp.Eup.Het.xx.Xga1. heterophylla. [228] Evonymus — —LECp.Evo.Eur.se.Hcu1. europaea. [229] Falcata japonica. — —LECp.Fal.Jap.se.Hga1. [230] Ficus cunia. — — LECp.Fic.Cun.xx.Xxxx. [231]Flammulina — — LECf.Fla.Vel.xx.Xxxx. veltipes. [232] Fomes — —LECz.Fom.Fom.xx.Xxxx. fomentarius. [233] Fragaria vesca. — —LECp.Fra.Ves.xx.Xxxx. [234] Fucus serratus. — — LECu.Fuc.Ser.xx.Xxxx.[235] Fucus vesiculosis. — — LECu.Fuc.Ves.xx.Xxxx. [236] Galactiatashiroi. — — LECp.Gal.Tas.se.Hga1. [237] Galactia — —LECp.Gal.Ten.se.Hga1. tenuiflora. [238] Galanthus nivalis. — Monocotlectin. LECp.Gal.Niv.bu.Hma1. [239] Galleria — — LECi.Gal.Mel.xx.Xxxx.mellonella. [240] Gallus gallus. GGL. S-lectin or GLTa.Gal.Gal.xx.Xxxx.Galectin. [241] Gallus gallus. Chicken Hepatic — LECa.Gal.Gal.xx.Xxxx.lectins (CHL). [242] Gallus gallus. Chicken egg — LECa.Gal.Gal.xx.Xxxx.agglutinins. [243] Gallus gallus. Chick Beta- S-lectin orLECa.Gal.Gal.xx.Xxxx. galactoside-Binding Galectin. lectins. [244]Gallus gallus. Chicken Serum C-lectin or LECa.Gal.Gal.xx.Xxxx.Mannose-Binding Collectin. Protein. [245] Gallus gallus. Chicken LiverC-lectin or LECa.Gal.Gal.xx.Xxxx. Mannose-Binding Collectin. Protein.[246] Gallus gallus. Chicken Thymic S-lectin or GLTa.Gal.Gal..xx.Xxxx.Electrolectin (CTE). Galectin. [247] Gallus gallus. Chick EmbryonicS-lectin or GLTa.Gal.Gal.xx.Xxxx. Skin Lectins. Galectin. [248]Genypterus — — LECi.Epi.Tre.xx.Xxxx. blacodes. [249] Geodia cydonium. —— LECi.Geo.Cyd.xx.Xga1. [250] Giardia lambia Taglin. —LECu.Gia.Lam.xx.Xxxx. Surface lectin. [251] Gliricida sepium. Lectin A,Lectin B. — LECp.Gli.Sep.se.Hga1 (Lectin A) LECp.Gli.Sep.se.Hga2 (LectinB). [252] Glossina — — LECi.Glo.Lon.xx.Xxxx. longipennis lectin. [253]Glycine max. SBA. Legume lectin. LECp.Gly.Max.se.Hga1. [254] Gonatanthus— — LECz.Gon.Pum.ti.Hcu1. pumilus. [256] Grateulopia — —LECu.Gra.Fil.xx.Xxxx. filicina. [257] Griffithsia — —LECu.Gri.Flo.xx.Xxxx. flosculosa. [258] Griffonia GS-I-A4, GS-I-A4,Legume lectin. LECp.Gri.Sim.se.Hga1 simplicifolia GS-I-B4, GS-II, GS-(GS-I-A4) lectins. IV. LECp.Gri.Sim.se.Hga2 (GS-I-B4)LECp.Gri.Sim.se.Hch1 (GS-II) LECp.Gri.Sim.se.Hfu1 (GS- IV)LECp.Gri.Sim.le.Hga1 (GS-I-A4) LECp.Gri.Sim.le.Hga2 (GS- I-B4)LECp.Gri.Sim.le.Hch1 (GS- II) LECp.Gri.Sim.le.Hfu1 (GS-IV). [260]Grifola frondosa. GFL. — LECf.Gri.Fro.xx.Xga1. [261] Haemonchus — —LECz.Xxx.Xxx.xx.Xxxx. contortus. [262] Halidrys siliquosa. — —LECu.Hal.Sil.xx.Xxxx. [263] Halimeda opuntia. — — LECu.Hal.Opu.xx.Xxxx.[264] Halocynthia — — LECi.Hal.Pyr.xx.Xxxx. pyriformis. [265]Halocynthia — — LECi.Hal.Ror.xx.Xga1. roretzi. [266] Haynaldia villosa.— Hevein domain LECp.Hay.Vil.se.Hch1. lectin, chitin binding. [269]Helianthus annus. — beta-prism plant LECp.Hel.Ann.xx.Xxxx. lectin. [270]Helianthus HTA. Jacalin-related LECp.Hel.Tub.tu.Hmmm1. tuberosus.lectins. [271] Helicobacter HP-SAL. — LECb.Hel.Pyl.xx.Xxxx. pylori.[272] Helix aspersa. — — LECi.Hel.Asp.xx.Xxxx. [273] Helix pomatia. HPA.— LECi.Hel.Pom.xx.Xxxx. [274] Herpetomonas. — — LECz.Her.xx.Xxxx. [276]Heteranthelium — Hevein domain LECp.Het.Pil.se.Hch1. piliferum. lectin,chitin binding. [277] Heterometrus — — LECi.Het.gra.xx.Xsi1.granulomanus. [279] Hevea brasiliensis. HBA, Hevein. Chitin-bindingLECp.Hev.Bra.la.Mch1. lectin with hevein domain. [280] Hippeastrum HHA.Monocot lectin. LECp.Hip.Hyb.bu.Hma1. hybrid. [281] Hippopus Tridacnin.C-lectin. LECi.Hip.Hip.xx.Xxxx. hippopus. [282] Hizoctonia solani. — —LECz.Hiz.Sol.xx.Xxxx. [283] Hohenbuehelia — — LECf.Hoh.Ser.xx.Xxxx.serotina. [284] Homarus HAA. — LECi.Hom.Ame.xx.Xxxx. americanus. [285]Homo sapiens. P-selectin (1KJD). C-lectin. LECh.Hom.Sap.xx.Xxxx. [286]Homo sapiens. Human Mannose C-lectin. LECh.Hom.Sap.xx.Xxxx. BindingProtein (MBP) (1HUP). [287] Homo sapiens. Gut Mucus Anti- —LECh.Hom.Sap.xx.Xxxx. Salmonella Lectin. [288] Homo sapiens. HumanMembrane — LECh.Hom.Sap.Xxxx. Lectins (HKML, HCCML). [289] Homo sapiens.Human Synovial — LECh.Hom.Sap.xx.Xxxx. Tissue Lectins. [290] Homosapiens. Human Placenta — LECh.Hom.Sap.xx.Xxxx. Lectins (HPL-H, HPL-BG).[291] Homo sapiens. Human Brain — LECh.Hom.Sap.xx.Xxxx.Galactoside-binding Lectin. [292] Homo sapiens. Human 14-kDa —LECh.Hom.Sap.xx.Xxxx. Lectins. [293] Homo sapiens. Human Core-specific —LECh.Hom.Sap.xx.Xxxx. Lectin (HCSL). [294] Homo sapiens. Cell Membrane —LECh.Hom.Sap.xx.Xxxx. Lectins. [295] Homo sapiens. Tumoricidal —LECh.Hom.Sap.xx.Xga1. Macrophage Lectin. [296] Homo sapiens.Tumor-associated — LECa.Ggg.Sss.xx.Xxxx. Vertebrate Lectin. [297] Homosapiens. Human Conglutinin- — LECh.Hom.Sap.xx.Xxxx. like Protein. [298]Homo sapiens. Mannose-Specific — LECh.Hom.Sap.xx.Xma1. EndocytosisReceptor. [299] Homo sapiens. Human Penultimate — LECh.Hom.Sap.xx.Xxxx.Galactose Lectin. [300] Homo sapiens. Thrombospondin. —LECh.Hom.Sap.xx.Xxxx. [301] Homo sapiens. Tetranectin. —LECh.Hom.Sap.xx.Xxxx. [302] Homo sapiens. Human Dendritic —LECh.Hom.Sap.xx.Xxxx. Cell Immunoreceptor (DCIR). [303] Homo sapiens.Human Seminal — LECh.Hom.Sap.xx.Xxxx. Lectin (HSL). [304] Homo sapiens.Charcot-Leyden S-lectin or GLTh.Hom.Sap.xx.Xxxx. crystal proteinGalectin. (1LCL). [305] Homo sapiens. Galectin II L-14-II Proto S-lectinor GLTh.Hom.Sap.xx.Xxxx. (1HLC). Galectin. [306] Homo sapiens. HumanLung C-lectin or GLTh.Hom.Sap.xx.Xxxx. Surfactant Protein Collectin.(1B08). [307] Homo sapiens. Galectin III. Chimera S-lectinGLTh.Hom.Sap.xx.Xxxx. or Galectin. [308] Homo sapiens. Galectin VII,hGal-7. Proto S-lectin or GLTh.Hom.Sap.xx.Xxxx. Galectin. [309] Homosapiens. Pentraxin (1CRV). Pentraxin, S- GLTh.Hom.Sap.xx.Xxxx. lectin orGalectin. [310] Homo sapiens. Sialoadhesin. I-lectin.LECz.Ggg.Sss.xx.Xxxx. [311] Homo sapiens. Serum Amyloid P Pentraxin.LECh.Hom.Sap.xx.Xxxx. Component. [312] Homo sapiens. E-Selectin (1ESL).C-lectin. SELh.Hom.Sap.xx.Xxxx. [313] Homo sapiens. L-Selectin (1KJB).C-lectin. SELh.Hom.Sap.xx.Xxxx. [314] Homo sapiens. C-Reactive proteinPentraxin, S- GLTh.Hom.Sap.xx.Xxxx. (1CRV). lectin or Galectin. [315]Homo sapiens. Galectin XII. S-lectin or GLTh.Hom.Sap.xx.Xxxx. Galectin.[316] Homo sapiens. Galectin I. Proto S-lectin or GLTh.Hom.Sap.xx.Xxxx.Galectin. [317] Homo sapiens. Galectin IX, Tandem RepeatGLTh.Hom.Sap.sr.Xxxx. Ecalectin. S-lectin or Galectin. [318] Homosapiens. Galectin VIII. Tandem Repeat GLTh.Hom.Sap.xx.Xxxx. S-lectin orGalectin. [319] Homo sapiens. Galectin IV. Tandem RepeatGLTh.Hom.Sap.xx.Xxxx. S-lectin or Galectin. [320] Homo sapiens.Alpha-1/Beta-1 Integrin A (or I) INTh.Hom.Sap.xx.Xxxx. integrin. domain.[321] Homo sapiens. Alpha-2/Beta-1 Integrin A (or I)INTh.Hom.Sap.xx.Xxxx. integrin. domain. [322] Homo sapiens.Alpha-3/Beta-1 Integrin A (or I) INTh.Xxx.Xxx.xx.Xxxx. integrin. domain.[323] Homo sapiens. Alpha-4/Beta-1 Integrin A (or I)INTh.Xxx.Xxx.xx.Xxxx. integrin. domain. [338] Homo sapiens.Alpha-5/Beta-8 Integrin A (or I) INTh.Hom.Sap.xx.Xxxx. integrin. domain.[339] Homo sapiens. Alpha-4/Beta-7 Integrin. INTh.Hom.Sap.xx.Xxxx.Integrin. [340] Homo sapiens. Alpha-E/Beta-7. Integrin.INTh.Hom.Sap.xx.Xxxx. [341] Homo sapiens. Mucosal addressin Addressin.LECh.Xxx.Xxx.xx.Xxxx. cell adhesion molecule-1 (MADCAM-1). [342] Homosapiens. Vascular Adhesion — LECh.Xxx.Xxx.xx.Xxxx. Molecule (VCAM-1.[343] Homo sapiens. P-Selectin Selectin. SELh.Xxx.Xxx.xx.Xxxx. [344]Homo sapiens. Intercellular Addressin?. LECh.Xxx.Xxx.xx.Xxxx. AdhesionMolecule (ICAM-1, ICAM-2). [345] Homo sapiens. Peripheral LymphAddressin. LECh.Xxx.Xxx.xx.Xxxx. Node Addressin (PNAd). [346] Homosapiens. Vascular Adhesion — LECh.Xxx.Xxx.xx.Xxxx. Protein (VAP-1).[347] Homo sapiens. LFA-3. Addression?. LECh.Xxx.Xxx.xx.Xxxx. [348] Homosapiens. Versican. Soluble C-lectin LECh.Xxx.Xxx.xx.Xxxx. (‘Lecticans’).[349] Homo sapiens. Aggrecan. Soluble C-lectin LECh.Xxx.Xxx.xx.Xxxx.(‘Lecticans’). [350] Homo sapiens. Neurocan. Soluble C-lectinLECh.Xxx.Xxx.xx.Xxxx. (‘Lecticans’). [351] Homo sapiens. Brevican.Soluble C-lectin LECh.Xxx.Xxx.xx.Xxxx. (‘Lecticans’). [352] Homosapiens. Annexin V. Annexin. ANNh.Hom.Sap.xx.Xxx5. [353] Homo sapiens.Annexin II. Annexin. ANNh.Hom.Sap.xx.Xxx2. [354] Homo sapiens. AnnexinIV. Annexin. ANNh.Hom.Sap.xx.Xxx4. [355] Homo sapiens. Annexin IAnnexin. ANNh.Hom.Sap.xx.Xxx1. (Lipocortin-1), ANX1. [356] Homo sapiens.Annexin VII, — ANNh.Hom.Sap.xx.Xxx7. Synexin. [357] Homo sapiens.Activated Leukocyte — LECh.Hom.Sapxx. Adhesion Molecule (ALCAM). [358]Homo sapiens. E-cadherin. — CDHh.Hom.Sap.xx.XxxE. [360] Homo sapiens.N-cadherin — CDHh.Hom.Sap.xx.XxxN. (uvomorulin). [361] Homo sapiens.VE-cadherin — CDHh.Hom.Sap.xx.XxxVE. (Vascular Endothelial Cadherin).[362] Homo sapiens. P-cadherin. — CDHh.Hom.Sap.xx.XxxP. [363] Homosapiens. Annexin XI (CAP- — ANNh.Hom.Sap.xx.Xxx9. 50). [364] Homosapiens. Endothelial Cell- — CDHh.Hom.Sapxxx. Selective AdhesionMolecule (ESAM). [365] Homo sapiens. ELAM-1. — CDHh.Xxx.Xxx.xx.Xxxx.[366] Homo sapiens. GMP-140. — CDHh.Xxx.Xxx.xx.Xxxx. [367] Homo sapiens.Cutaneous — LECh.Xxx.Xxx.xx.Xxxx. Lymphocyte Antigen (CLA). [369] Homosapiens. Lymphocyte — LECh.Xxx.Xxx.xx.Xxxx. Function-AssociatedAntigen-1 (LFA-1). [370] Homo sapiens. Very Late Antigen 4 —LECh.Xxx.Xxx.xx.Xxxx. (VLA-4). [371] Hordeum vulgare. HVA. —LECp.Hor.Vul.se.Hch1. [372] Hura crepitans. HCA. Type 2 RIP.LECp.Hur.Cre.se.Cga1 (HCA) LECp.Hur.Cre.la.Cga1. [373] Hygrophorus — —LECf.Hyg.Hyp.xx.Xxxx. hypothejus. [374] Hypnea — — LECu.Hyp.Cer.xx.Xxxx.cervicornis. [375] Hyptos — — LECz.Hyp.Sua.xx.Xxxx. suaveolens. [376]Iberis amara. — — LECp.Ibe.Ama.xx.Xxxx. [377] Influenza virus.Hemagglutinin. Hemagglutinin. LECv.Inf.Vir.xx.Xxxx. [378] Ipomoeabatatas. — — LECp.Ipo.Bat.xx.Xxxx. [379] Iris hollandica. — —LECp.Iri.Hol.xx.Xxxx. [380] Iris hybrid. IRA. Type 2 RIP.LECp.Iri.Hyb.bu.Cga1. [381] Juglans regia. — — LECp.Jug.Reg.xx.Xxxx.[382] Klyveromyces — — LECz.Kly.Bul.xx.Xxxx. bulgaricus. [383]Kuehneromyces — — LECu.Kue.Mut.xx.Xxxx. mutabilis. [384] Labiaceae — —LECp.Lab.Ori.xx.Xxxx. origanum. [385] Lablab purpureus. DLA, LPA. Legumlectin. LECp.Lab.Pur.se.Hmg1. [386] Laburnum LAA-I, LAA-II. Legumelectin. LECp.Lab.Alp.se.Hch1 alpinum. (LAA-I) LECp.Lab.Alp.se.Hga1(LAA-II). [387] Laccaria — — LECz.Lac.Ame.xx.Xxxx. amethystina. [389]Lachesis huta. BML. — LECi.Lac.Jut.xx.Xxxx. [390] Lactarius LDL. —LECf.Lac.Del.xx.Xgal1. deliciosus. [391] Lactarius — —LECz.Lac.Lig.xx.Xxxx. lignyotus. [392] Lactuca scariole. PLA-I, PLA-II.— LECa.Lac.Sca.xx.Xxxx. [393] Laelia autumnalis. — —LECp.Lae.Aut.xx.Xxxx. [394] Laetiporus PSL. — LECf.Lae.Sul.xx.Xxxx.sulfureus. [395] Lathyrus cicera. LcLI, LcLII. — LECp.Lat.Cic.xx.Xxxx.[396] Lathyrus nissolia. — — LECp.Lat.Nis.xx.Xxxx. [397] Lathyrusochrus. LOL-I, LOL-II. Legume lectin. LECp.Lat.Och.xx.Xxxx. [398]Lathyrus odoratus. — — LECp.Lat.Odo.xx.Xxxx. [399] Lathyrus silvestris.— — LECp.Lat.Sil.xx.Xxxx. [400] Lathyrus — — LECp.Lat.Tub.xx.Xxxx.tuberosus. [401] Lens culinaris. LCA, LcH. Legume lectins.LECp.Len.Cul.se.Hmg1. [402] Lepidium — — LECp.Lep.Sat.xx.Xxxx. sativuum.[403] Leptonychotes — — LECz.Lep.Wed.xx.Xxxx. weddelli. [404]Leptospermum LAA. — LECp.Lep.Arc.xx.Xxxx. archinoides. [405] Leucojum. —— LECz.Leu.sss.xx.Xxxx. [406] Leucojum LAA. MonocotLECp.Leu.Aes.bu.Hma1. aestivum. mannose-binding lectins. [407] Leucojumvernum. LVA. Monocot LECp.Leu.Ver.bu.Hma1. mannose-binding lectins.[408] Limulus Limulin. Pentraxin. LECi.Lim.Pol.xx.Xsi1. polyphemus.[409] Liocarcinus — — LECi.Lio.Dep.xx.Xxxx. depurator. [410] Listeriaovata. LOA, LOMBP. Monocot LECp.Lis.Ova.le.Hma1 mannose binding (LOA)proteins. LECp.Lis.Ova.le.Mma1 (LMOBP). [411] Litchi chinensis. LCL. —LECp.Lit.Chi.xx.Xxxx. [412] Lonchocarpus — Legume lectin.LECp.Lon.Cap.se.Hga1. capassa. [413] Lontonis bainesii. — Legum lectins.LECp.Lon.Bai.se.Hga1 LECp.Lon.Bai.ro.Hga1. [414] Lophocereus — —LECp.Lop.Sho.xx.Xxxx. shotti. [415] Lotus LTA. Legume lectins.LECp.Lot.Tet.se.Hfu1. tetragonolobus. [416] Luffa acutangula. LAA.Cucurbtaceae LECp.Luf.Acu.fr.Hch1. phloem lectins. [417] Lumbricus EW29.— LECi.Lum.Ter.xx.Xxxx. terrestris. [418] Lycopersion LEA, TL, LEL.Chitin-binding LECp.Lyc.Esc.fr.Hch1. esculentum. lectins. [419] Lycorisaurea. — Monocot LECp.Lyc.Aur.bu.Hma1. mannose-binding lectins. [420]Maackia MALb, MAHb, Legume lectins. LECp.Maa.Amu.se.Hsi1 amurensis. MAL,MAHs. (MAHs, MAH) LECp.Maa.Amu.se.Hsi2 (MAHs, MAH) LECp.Maa.Amu.ba.Hsi1(MAHb) LECp.Maa.Amu.ba.Hsi1 (MALb). [421] Machaerocereus MEA-I, MEA-II.?. LECp.Mac.Eru.st.Hga1. eruca. [422] Machaerocereus — Hevein domainLECp.Mac.Gum.xx.Xxxx. gummosus. lectin, chitin binding. [423] Maclurapomifera. MPA. beta-prism plant LECi.Mac.Pom.xx.Xxxx. lectin. [424]Macrobdella LL1-63. — LECi.Mac.Dec.xx.Xxxx. decora. [425] MacrobrachiumMrL. — LECi.Mac.Ros.xx.Xxxx. rosenbergii. [426] Macrotyloma — —LECp.Mac.Axi.xx.Xxxx. axillare. [427] Malus officinalis. — —LECp.Mal.Off.xx.Xxxx. [428] Manduca sexta. Immulectin. C-lectin.LECi.Man.Sex.xx.Xxxx. [429] Mangifera indica. MIA. —LECp.Man.Ind.xx.Xxxx. [430] Marah — — LECp.Mar.Mac.xx.Xxxx. macrocarpus.[431] Marasmius — — LECf.Mar.Ore.xx.Xxxx. oreades. [432] Medicagosativa. — — LECp.Med.Sat.xx.Xxxx. [433] Medicago — —LECp.Med.Tru.xx.Xxxx. truncatula. [434] Megabalanus rosa. — —LECi.Mag.Ros.xx.Xxxx. [435] Megapitaria — — LECi.Meg.Squ.xx.Xxxx.squalida. [436] Melanoleuca — — LECf.Mel.Mel.xx.Xxxx. melaleuca. [437]Melastiza chateri. — — LECf.Mel.Cha.xx.Xxxx. [438] Mesocricetus —Pentraxin. LECz.Ggg.Sss.xx.Xxxx. auratus. [474] Orchidaceae. — —LECp.Orc.Sss.xx.Xxxx. [475] Ornithodoros Dorin-M. —LECi.Orn.Mou.xx.Xxxx. moubata. [476] Oryza sativa. OSA. Chitin bindingLECp.Ory.Sat.see.Hch1. lectins. [477] Oscillatoria — —LECu.Osc.Aga.xx.Xxxx. agardhii. [478] Otala lactea. — —LECi.Ota.Lac.xx.Xxxx. [480] Pachydereus — — LECp.Pac.Pri.se.Hch1.pringleii. [481] Pacifastacus — — LECi.Pac.Len.xx.Xxxx. leniusculus.[482] Palmaria palmata. — — LECz.Pal.Pal.xx.Xxxx. [483] Paracentrotus —— LECi.Par.Liv.xx.Xxxx. lividus. [484] Parkia — — LECp.Par.Big.xx.Xxxx.biglandulosa. [485] Parkia discolor. — — LECz.Par.Dis.xx.Xxxx. [486]Parkia — — LECz.Par.Pla.xx.Xxxx. platycephala. [487] Parkia speciosa. —— LECp.Park.Spe.xx.Xxxx. [488] Paxillus — — LECz.Pax.Atr.xx.Xxxx.atrotomentosus. [489] Paxillus — — LECf.Pax.Pan.Sss.xx.Xxxx. panuoides.[490] Penaeus — — LECp.Pen.Cal.xx.Xxxx. californiensis. [492] Penaeus —— LECi.Pen.Sty.xx.Xxxx. stylirostris. [494] Penaeus vannamei. — —LECi.Pen.Van.xx.Xxxx. [495] Perca fluviatilis. — — LECa.Per.Flu.xx.Xxxx.[496] Peresea gratissima. — — LECz.Per.Gra.xx.Xxxx. [497] Perseaamericana. PAA. — LECp.Per.Ame.xx.Xxxx. [498] Petromyzon — —LECz.Pet.Mar.xx.Xxxx. marinus. [499] Petrosecinum — —LECp.Pet.Hor.xx.Xxxx. hortense. [500] Peziza badia. — —LECp.Pez.Bad.xx.Xxxx. [501] Phage p22. Phage P22 — LECb.Ggg.Sss.xx.Xxxx.TailspikeProteins (1TSP). [502] Phalera flavescens. PFA. —LECi.Pha.Fla.xx.Xxxx. [503] Phallus impudicus. — — LECf.Pha.Imp.xx.Xxxx.[504] Phallusia — — LECi.Pha.Mam.xx.Xxxx. mamillata. [505] Phaseolus —Legume lectins. LECp.Pha.Acu.se.Hcu1 acutifolius. (erythroagglutinin)LECp.Pha.Acu.se.Hcu2 (lymphoagglutinin). [506] Phaseolus aureus. — —LECp.Pha.Aur.xx.Xxxx. [507] Phaseolus PCA. Legume lectin.LECp.Pha.Coc.se.Hcu1 coccineus. (PCA) LECp.Pha.Coc.se.Hcu2. [508]Phaseolus — — LECp.Pha.Coc.xx.Xxxx. coccineus. [509] Phaseolus PLA, LBA,LBL. Legume lectins. LECp.Pha.Lim.se.Hga1. limenesis. [510] Phaseoluslunatus. — — LECp.Pha.Lun.se.Xxxx. [511] Phaseolus PHA-E, PHA-L. Legumelectin. LECp.Pha.Vul.xx.Xxxx. vulgaris. [512] Phaseolus GNlL, GNpL,GNsL. — LECp.Pha.Vul.xx.Xxxx. vulgaris. [513] Phaseolus Pinto III. —LECa.Pha.Vul.xx.Xxxx. vulgaris. [514] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [515] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [516] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [517] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [518] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [519] Phaseolus — —LECp.Pha.Vul.xx.Xxxx. vulgaris. [520] Phlomis — — LECz.Phl.Fru.xx.Xxxx.fructicosa. [521] Pholiota aurivella. PAA. — LECf.Ggg.Sss.xx.Xxxx. [522]Pholiota squarrosa. — — LECf.Pho.Squ.xx.Xxxx. [524] Phoradendron — —LECz.Pjo.Cal.xx.Xxxx. californicum. [525] Phragmites. — —LECz.Phr.sss.xx.Xxxx. [526] Phragmites — — LECp.Phr.Aus.xx.Xxxx.austalis. [527] Physalia physalis. Physalitoxin. — LECi.Phy.Phy.xx.Xxxx.[528] Physalis angulata. PA-VII-A, PA-VII-B — LECz.Phy.Ang.xx.Xxxx. andPA-VII-C. [529] Physarum — — LECu.Phy.Pol.xx.Xxxx. polycephalum. [530]Phytolacca PWM, Pa-1, Pa-2 — LECp.Phy.Ame.ro.Hch1 (Pa- americana. (PL-A)Pa-3, Pa-4 1) (PL-C), Pa-5. LECp.Phy.Ame.ro.Hch2(Pa- 2)LECp.Phy.Ame.ro.Hch3(Pa- 3) LECp.Phy.Ame.ro.Hch4(Pa- 4)LECp.Phy.Ame.ro.Hch5(Pa- 5) LECp.Phy.Ame.ro.Hch6(PL- B). [531] Pimenta —— LECp.Pim.Off.xx.Xxxx. officinalis. [532] Pisum sativum. PSA, PsA.Legume lectins. LECp.Pis.Sat.se.Hmg1 (PSA, PsA) LECp.Pis.Sat.ro.Hmg1.[533] Plecoglossus PAL. — LECa.Ple.Alt.xx.Xxxx. altivelis. [535]Pleurocybella — — LECf.Ggg.Sss.xx.Xxxx. porrigens. [536] Pleurotus — —LECf.Ple.Ost.xx.Xxxx. ostreatus. [537] Plumaria elegans. — —LECu.Plu.Ele.xx.Xxxx. [538] Polyandrocarpa — C-lectin.LECi.Pol.Mis.xx.Xgal. misakiensis. [539] Polygonum — —LECp.Pol.Mul.xx.Xxxx. multiformum. [540] Polyomavirus. 1VPN. —LECV.Pol.Vir.xx.Xxxx. [541] Polyporus — — LECf.Pol.Fom.xx.Xxxx.fomentarius. [542] Polyporus — — LECf.Pol.Squ.xx.Xxxx. squamosus. [543]Polysphondylium — — LECu.Pol.Pal.xx.Xxxx. pallidum. [544] Potamon — —LECi.Pot.Pot.xx.Xxxx. potamios. [545] Prunus americana. — —LECp.Pru.Ame.xx.Xxxx. [546] Prunus avium. — — LECp.Pru.Avi.xx.Xxxx.[547] Psathyrostachys — Hevein domain LECp.Psa.Jun.se.Hch1. juncea.lectin, chitin binding. [548] Pseudomonas — — LECb.Pse.Aer.xx.Xga1.aeruginosa. [549] Pseudomonas — — LECb.Pse.Apl.xx.Xxxx. aplysia. [550]Psophocarpus PTL-I (WBA-I), — LECp.Pso.Tet.se.Hga1 tetragonolobus.PTL-II (WBA-II), (PTL-I) WBTL, L-I, L-II. LECp.Pso.Tet.se.Hga2 (PTL-II)LECp.Pso.Tet.ro.Hga1 (WBTL) LECp.Pso.Tet.so.Hga2 LECp.Pso.Tet.le.Hga1(L-I) LECp.Pso.Tet.le.Hga2 (L- II). [551] Ptilota serrata. — —LECu.Pxx.Ser.xx.Xxxx. [552] Punica granatum. — — LECp.Pun.Gra.xx.Xxxx.[553] Rana catesbeiana. — Lectins LECa.Ran.Cat.xx.Xxxx. Displaying RNaseActivity (Leczymes). [554] Rana catesbeiana cSBL. LectinsLECa.Ran.Cat.xx.Xxxx. ovum lectin. Displaying RNase Activity (Leczymes).[555] Rana japonica. jSBL. Lectins LECa.Ran.Jap.xx.Xxxx. DisplayingRNase Activity (Leczymes). [557] Rana — — LECa.Ran.Nig.xx.Xxxx.nigromaculata. [558] Raphanus sativus. — — LECp.Rap.Sat.xx.Xxxx. [559]Ratus norvegicus. Mannan Binding C-lectin or LECa.Rat.Nor.xx.Xxxx.Protein (MBP-A). Collectin. [560] Ratus ratus. Rat peritioneal —LECa.Rat.Rat.xx.Xfu1 macrophage lectin. LECa.Rat.Rat.xx.Xga1. [561]Ratus ratus. — — LECa.Rat.Rat.xx.Xxxx. [562] Ratus ratus. Galectin II.S-lectin or GLT2.Rat.Rat.xx.Xxxx. Galectin. [563] Ratus ratus. GalectinIV. Tandem Repeat GLTa.Rat.Rat.xx.Xxxx. S-lectin or Galectin. [564]Rheum — — LECp.Rhe.Rhas.xx.Xxxx. rhapontium. [565] Ribes rubrum. — —LECp.Rib.Rubs.xx.Xxxx. [566] Ricinus RCA-I, RCA-II, beta-trefoil lectin.LECp.Ric.Com.se.Cga1 communis. Ricin. (Ricin D) LECp.Ric.Com.se.Cga2(Ricin E) LECp.Ric.Com.se.Cga2 (RCA, RSL). [567] Robinia RPA-I, RCA-III,— LECp.Rob.Pse.se.Hcu1 pseudoacacia. (RPsA-I) LECp.Rob.Pse.se.Hcu2(RPsA-II) LECp.Rob.Pse.se.Hcu1 (RPbA-I) LECp.Rob.Pse.se.Hcu2 (RPbA-II).[568] Rubus fructicosus. RFA. ?. LECp.Rub.Fru.tc.Xga1. [569] Rubusidaeus. — — LECp.Rub.Ida.xx.Xxxx. [570] Rutilus rutilus. — —LECv.Rut.Rut.xx.Xxxx. [571] Salmo gairdneri. — — LECa.Sal.Gai.xx.Xxxx.[572] Salmo salar v. — — LECa.Sal.Sal.xx.Xma1. atlantica. [573] Salmosalar v. — — LECa.Sal.Sal.xx.Xxxx. chinook. [574] Salmo trutta. — —LECa.Sal.Tru.xx.Xxxx. [618] Tetragonolobus — — LECp.Tet.Pur.xx.Xxxx.pupurea. [619] Thermopsis. — — LECz.The.sss.xx.Xxxx. [621] Toxopneustes— C-lectin. LECi.Xxx.Xxx.xx.Xxxx. pileolu. [622] Trichoderma. — —LECf.Tri.Sss.xx.Xxxx. [623] Tricholoma — — LECf.Tri.Mon.xx.Xxxx.mongolicum. [624] Tricholomataceae — — LECf.Tri.Sss.xx.Xxxx. 93-138.[625] Tricholomataceae — — LECz.Tri.Sss.xx.Xxxx. 93-34. [626]Trichosanthes TJA-II, TJA-I, TK-I, — LECp.Tri.Jap.xx.Xxxx. japonica.TK-II. [627] Trifolium repens. — — LECp.Tri.Rep.xx.Xxxx. [628] TriticumWGA. Hevein domain LECp.Tri.Aes.se.Hch1. aestivium. lectin, chitinbinding lectin. [629] Tulipa gesneriana. TGA. — LECp.Tul.Ges.xx.Xxxx.[630] Udotea petiolata. — — LECp.Udo.Pet.xx.Xxxx. [631] Ulex europaeus.UEA-I, UEA-II, Legume lectin. LECp.Ule.Eur.xx.Xxxx. UEA-III. [632] Ulvalactuca. — — LECu.Ulv.Lac.xx.Xxxx. [633] Ulva laetevirens. — —LECu.Ulv.Lae.xx.Xxxx. [635] Ulva rigida. — — LECz.Ulv.Rig.xx.Xxxx. [637]Urtica dioica. UDA. Chitin-binding LECp.Urt.Dio.rh.Hch1. lectins. [638]Vaejovis — — LECi.Vae.Con.xx.Xxxx. confuscius. [639] Vatairea VML. —LECp.Vat.Mac.xx.Xxxx. macrocarpa. [640] Vibrio — — LECb.Vib.Alg.xx.Xch1.alginolyticus. [641] Vibrio cholera. VPCV; Chitovibrin —LECb.Vib.Cho.xx.Xxxx. [642] Vicia cracca. — — LECp.Vic.Cra.xx.Xxxx.[643] Vicia ervilia. — — LECp.Vic.Erv.xx.Xxxx. [644] Vicia faba. VFA,Favin. Legume lectin. LECp.Vic.Fab.xx.Xxxx. [645] Vicia graminea. VGA. —LECp.Vic.Gra.xx .Xxxx. [646] Vicia hyrcanica. — — LECp.Vic.Hyr.xx.Xxxx.[647] Vicia sativa. — — LECp.Vic.Sat.xx.Xxxx. [648] Vicia unijuga. VUA.— LECp.Vic.Unj.xx.Xxxx. [649] Vicia villosa. VVA-A4, VVL-A4. Legumelectin. LECp.Vic.Vil.xx.Xxxx. [650] Vigna radiata. MBL-I, MBL-II. —LECp.Vig.Rad.xx.Xxxx. [651] Vigna unguiculata. — — LECp.Vig.Ung.xx.Xxxx.[652] Viscum album. ML-I, ML-II, ML-III, Beta-trefoil lectinLECp.Vis.Alb.pl.Cga1 Viscumin, (ML-I). (ML-I, viscumin) VisAlbCBA.LECp.Vis.Alb.pl.Cga2 (ML-II, viscumin) LECp.Vis.Alb.pl.Cga3 (ML-III,VAA-II) LECp.Vis.Alb.pl.Hch1 (VisAlbCBA). [653] Vitis vinifera. — —LECp.Vit.Vin.xx.Xxxx. [654] Volvariella VVL. — LECf.Vol.Vol.xx.Xxxx.volvacea. [655] Wistaria WFA. — LECp.Wis.Flo.xx.Xxxx. floribunda. [656]Wistaria — — LECp.Wis.Flo.xx.Xxxx. floribunda. [657] Wistaria sinensis.— — LECp.Wis.Sin.xx.Xxxx. [658] Wistaris — — LECz.Wis.Bra.xx.Xxxx.brachbotrys. [659] Xanthosoma — — LECp.Xan.Sag.xx.Xxxx. sagittifolium.[660] Xenopus laevis — — LECa.Xen.Lae.xx.Xga1. ovum. [661] Xeromus — —LECz.Xer.Chr.xx.Xxxx. chrysenteron. [662] Xylaria — —LECf.Xyl.Pol.xx.Xxxx. polymorpha. [663] Zea mays. ZMA-I, ZMA-II, —LECp.Zea.May.xx.Xxxx. ZMEA. [664] Cannabis sativa. CSA. —LECp.CanSat.se.Glu. [665] Smilax glabra. Sarparilla. —LECp.SmiGla.rh.xxx. [666] Trichosanthes Snake gourd. — — anguina.

[0114] Lectin codes take the following form: LLLx.Ggg.Sss.ti.TspN

[0115] An explanation of each index variable follows.

[0116] LLL refers to the general category of agglutinin. At this pointsix general categories are recognized: lectins (LEC), integrins (INT),cadherins (CDH), annexins (ANN), selectins (SEL) and galectins (GLT).The x value refers to the taxonomic groups of the agglutinin, Table 1summarizes these categories: Category Taxonomic group LECa, GLTa Lectinor galectin from higher animal, typically vertebrates. LECh, GLTh Lectinor galectin from humans LECi, GLTi Lectin or galectin from invertebratesLECp. Plant lectins LECf. Lectin from fungi LECu. Lectin fromunicellular organisms LECb. Lectin from Bacteria LECv. Viral lectins

[0117] Ggg stands for the three first letters of the plant genus name(in Latin).

[0118] Sss stands for the three first letters of the plant species name(in Latin).

[0119] ti refers to the tissue from which the lectin has been isolated.Table 2 summarizes the indices used for the various tissues: Tissue,cell or organ Taxonomic grouping Index Bark Plant Ba Bulb Plant Bu Cellmembrane Bacteria, Unicellular Cm Epidermis Human, vertebrates Ep FruitPlant Fr Hemolymph Invertebrates He Latex Plant La Leaf Plant Le NodulePlant No Organ or cell type Human, vertebrates, Oc Invertebrates Phloemsap Plant Ps Rhizome Plant Rh Root Plant Ro Seed Plant Se Serum orplasma Human, vertebrates, Sr Invertebrates Spores or fruiting bodiesFungi Sp Stem Plant St Tentacles Invertebrates Te Tuber Plant Tu Wholebody homogenate Invertebrates Wb Venom Invertebrates Ve Undefined Human,vertebrates, Un Invertebrates, Bacteria, Unicellular, Virus, Fungal

[0120] T refers to the lectin subtype. Hololectins, merolectins,chimerolectins and superlectins are indicated by the letters H, M, C andS, respectively.

[0121] sp refers to the specificity group. Each group is indicated bythe index given in TABLE 3 Specificity Index of group Mannose-bindinglectins ma Mannose/maltose-binding mm lectins Mannose/glucose-binding mglectins GlcNAc/(GlcNAc)-binding ch lectins Gal/GalNAc-binding lectins gaFucose-binding lectins fu Sialic acid-binding lectins si Lectins with acomplex but co known specificity Lectins with a complex and cu unknownspecificity Lectins with a dual specificity du Lectins with anundetermined nd specificity

[0122] Lipids

[0123] A multifunctional molecule of the invention can also be amolecule that comprises a first part which comprises a lipid and asecond part which comprises an amino acid sequence which can bind to acell surface molecule, e.g. a cell surface molecule of an APC. Theattachment of a lipid, e.g. a long-chain fatty acid, to a molecule, e.g.a polypeptide, can permit the complex to become stably associated withthe plasma membrane when the complex is admixed with a cell (Nagarajanet al, 1995, J Immunol Methods 184:241-51; McHugh et al, 1995, PNAS92:8059-63; van den Berg et al, 1995, J Cell Biol, 131:669-77). This isbelieved to occur through intercalation of the lipid into the membrane.A convenient method of producing a lipid-associated polypeptidecomprises expressing, in a suitable host cell, a nucleic acid encoding,in part, a signal sequence directing the post-translational addition ofa GPI moiety. Using recombinant DNA technology, a naturally non-GPIlinked protein can be expressed as a GPI-linked protein by constructinga nucleic acid that encodes the protein linked to a heterologous GPIsignal sequence. Nucleotide sequences encoding GPI signal sequencesuseful for this purpose include, for example, those comprised by decayaccelerating factor (e.g., sequences encoding amino acid sequence “22”in Table 1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta1292:223-32; sequences encoding signal sequences disclosed in Caras etal, U.S. Pat. No. 5,109,113); brevican (e.g., nt 1982-2047 of Genbankaccession number X86406), mesothelin (e.g., nt 1858-1983 of GenbankU40434), coccidioides immitis antigen 2 (e.g., sequences encoding aminoacids 172-194 of NCBI Entrez protein database accession # 1256444, Zhuet al, 1996, Gene 181:121-5), acetylcholinesterase (e.g., sequencesencoding the peptide “HC” as described in Duval et al, 1992, EMBO J11:3255-61; (e.g., sequences encoding amino acid sequence “19” in Table1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32)),human folate receptors alpha and beta (e.g., sequences encoding aminoacids 230-257 of NCBI Entrez protein database accession # 182416 oramino acids 228-255 of NCBI Entrez protein database accession # 1655592,Yan and Ratnam, 1995, Biochemistry 34:14594-600), 5′ nucleotidase (e.g.,sequences encoding amino acids 547-570 or 547-574 of NCBI Entrez proteindatabase accession # 404502, Furukawa et al, 1994, Biochim Biophys Acta1190:273-8; (e.g., sequences encoding amino acid sequences “5” or “6” inTable 1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta1292:223-32)), CD59 (e.g. encoded by nt 393-473 of Genbank U48255;sequences encoding amino acid sequence “20” in Table 1 of Bucht andHjalmarsson, 1996, Biochim Biophys Acta 1292:223-32; sequences encodingamino acids 74-101 of FIG. 2 of Powell et al, 1997, J Immunol158:1692-1702), T-cadherin (e.g., sequences encoding the 76 C-terminalamino acids of chick T cadherin as described by Koller and Ranscht,1996, J Biol Chem 271:30061-7), aminopeptidase P (e.g., sequencesencoding amino acids 649-673 of NCBI Entrez protein database accession #1517942, Hyde et al, 1996, Biochem J 319:197-201), carboxypeptidase M,CD16B, Thy 1, carbonic anhydrase IV (e.g., sequences encoding aminoacids 284-312 of NCBI Entrez protein database accession # 179791,Okuyama et al, 1995, Arch Biochem Biophys 320:315-22), placentalalkaline phosphatase (e.g., sequences encoding amino acids 498-529 ofNCBI Entrez protein database accession # 178464, Oda et al, 1994,Biochem J 301:577-83), neuronal glycoprotein F3, carcinoembryonicantigen (e.g., sequences encoding amino acid sequence “28” in Table 1 ofBucht and Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), MRC-OX45(e.g., sequences encoding amino acid sequence “2” in Table 1 of Buchtand Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), RT 6.2 (e.g.,sequences encoding amino acid sequence “3” in Table 1 of Bucht andHjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), D. discoideumprespore-specific antigen (e.g., sequences encoding amino acid sequence“4” in Table 1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta1292:223-32), microsomal dipeptidase (e.g., sequences encoding aminoacid sequence “8” in Table 1 of Bucht and Hjalmarsson, 1996, BiochimBiophys Acta 1292:223-32), CAMPATH-1 (e.g., sequences encoding aminoacid sequence “9” in Table 1 of Bucht and Hjalmarsson, 1996, BiochimBiophys Acta 1292:223-32), T. brucei PARP (e.g., sequences encodingamino acid sequence “10” in Table 1 of Bucht and Hjalmarsson, 1996,Biochim Biophys Acta 1292:223-32), T. brucei VSG Mit 118a (e.g.,sequences encoding amino acid sequence “11” in Table 1 of Bucht andHjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), T. brucei VSG Mit117a (e.g., sequences encoding amino acid sequence “12” in Table 1 ofBucht and Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), T.brucei VSG MITat 1.1000 BC (e.g., sequences encoding amino acid sequence“13” in Table 1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta1292:223-32), T. brucei VSG MITat 1.5b (e.g., sequences encoding aminoacid sequence “14” in Table 1 of Bucht and Hjalmarsson, 1996, BiochimBiophys Acta 1292:223-32), T. brucei VSG ILTat 1.1 (e.g., sequencesencoding amino acid sequence “15” in Table 1 of Bucht and Hjalmarsson,1996, Biochim Biophys Acta 1292:223-32), T. brucei VSG TxTat 1 (e.g.,sequences encoding amino acid sequence “16” in Table 1 of Bucht andHjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), T. brucei VSG Mit221 (e.g., sequences encoding amino acid sequence “17” in Table 1 ofBucht and Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32), prionproteins (e.g., sequences encoding amino acid sequence “18” in Table 1of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta 1292:223-32),urokinase receptor (e.g., sequences encoding amino acid sequence “21” inTable 1 of Bucht and Hjalmarsson, 1996, Biochim Biophys Acta1292:223-32), T. congolense VSG YNat 1.1 (e.g., sequences encoding aminoacid sequence “23” in Table 1 of Bucht and Hjalmarsson, 1996, BiochimBiophys Acta 1292:223-32), S. cerevesiae GAS-1 (e.g., sequences encodingamino acid sequence “24” in Table 1 of Bucht and Hjalmarsson, 1996,Biochim Biophys Acta 1292:223-32), Thy-1 (e.g., sequences encoding aminoacid sequences “25” or “26” in Table 1 of Bucht and Hjalmarsson, 1996,Biochim Biophys Acta 1292:223-32), L. major PSP (e.g., sequencesencoding amino acid sequence “29” in Table 1 of Bucht and Hjalmarsson,1996, Biochim Biophys Acta 1292:223-32), D. discoideum contact site Aglycoprotein (e.g., sequences encoding the 25 C-terminal amino acids asdescribed in Barth et al, 1996, Biochem J 317:533-40)CD24, and syntheticsequences (e.g. as described by Coyne et al, 1993, J Biol Chem268:6689-93).

[0124] GPI-linked polypeptides can be extracted from cells using thefollowing method. 5×10⁶ cells are spun down and frozen at −80° C. Thepellet is thawed in 14 ml of 0.15M NaCl/10 mM Tris 7.4/0.1 mMprimaquine/2% Trito X-114 with stirring at 0° C. for 1 h, thencentrifuged at 8800 g at 0° C. for 10 min. The supernatant is maintainedat −20° C. overnight, thawed at room temperature, and then placed at 32°C. for 12 min. It is then centrifuged at 3000 g for 3 min at 32° C. Thetop layer is decanted and 11 ml of cold Buffer A (0.15M NaCl/10 mM Tris7.4/0.1 mM primaquine/0.06% Triton X-114) is added to the bottom layer.This is incubated on ice for 10 min. The 12 min 32° C. incubation, 32°C. 3000 g centrifugation, decanting of top layer, and addition of 11 mlcold Buffer A to bottom layer are repeated. The solution is centrifugedat 18000 g for 10 min at 0° C. The 12 min 32° C. incubation, 32° C. 3000g centrifugation, and decanting of top layer are repeated. 3 vol of coldacetone are added to the final bottom phase. The solution is centrifugedat 12, 000 RPM for 30 min, the supernatant removed, and the proteinpellet containing the GPI fraction dried under vacuum. Specific proteinscan be purified by methods well-known to those skilled in the art, e.g.immunoaffinity purification.

[0125] Another method of producing a lipid-linked polypeptide is tochemically link the polypeptide to a fatty acid such as palmitate. 1.5mg/ml of the polypeptide is suspended in PBS, pH 7.8, containing 0.3%deeoxycholic acid, 0.1% sodium bicarbonate, and 0.1% sodium azide. Theoptimal final pH of the solution is 7.6-8.0. The mixture is warmed to37° C. and the N-hydroxysuccinimide ester of palmitic acid (ResearchOrganics, Cleveland, Ohio) is added to a final concentration of 0.1mg/ml. The solution is incubated overnight at room temperature. Thepolypeptide is purified by passage through a 16×250 mm Sephadex G-75chromatography column equilibrated with 0.15% deoxycholic acid in PBS,pH 7.6.

[0126] Crosslinking Moieties Useful According to the Invention

[0127] Another convenient method of linking a ligand to an antigenbearing target is to use a crosslinking agent. A “crosslinking agent” isa chemical entity that can react with functional groups on at least twoother molecules, e.g. two polypeptides or a polypeptide and a lipid,such that upon reaction with the crosslinking agent the two moleculesbecome covalently linked. Thus, a ligand for CD40 can be crosslinked toa molecule on the surface of a cell.

[0128] A wide variety of crosslinking agents, both bifunctional andpolyfunctional, are known in the art and are commercially available,e.g. from Sigma (St. Louis, Mo.). These include, for example,S-acetylmercaptosuccinic anhydride, S-acetylthioglycolic acidN-hydroxysuccinimide ester, S-acetylthiopropionic acidN-hydroxysuccinimide ester, adipic acid dihydrazide, 4-azidobenzoic acidN-hydroxysuccinimide ester, N-(5-azido-2-nitrobenzyloxy)succinimide,6-(4-azido-2-nitrophenylamino)hexanoic acid N-hydroxysuccinimide ester,p-azidophenacyl bromide, N-(4-azidophenylthio)phthalimide,4-azidosalicylic acid N-hydroxysuccinimide ester, bromoacetic acidN-hydroxysuccinimide ester, 1,4-butanediol diglycidyl ether,carbonyl-bis(L-methionine p-nitrophenyl ester),2-diazo-3,3,3-trifluoropropionic acid p-nitrophenyl ester, diethylmalonimidate, 1,5-difluoro-2,4-dinitrobenzene,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, dimethyladipimidate, dimethyl 3,3′-dithiobispropionimidate, dimethylpimelimidate, dimethyl suberimidate, 4,4′-dithiobisphenyl azide,dithiobis(propionic acid N-hydroxysuccinimide ester), ethylene glycolbis-(succinic acid N-hydroxysuccinimide ester), 4-fluoro-3-nitrophenylazide, bis-(4-fluoro-3-nitrophenyl) sulfone, p-formylbenzoic acidN-hydroxysuccinimide ester, glutaraldehyde, 2-iminothiolane,6-(iodoacetamido)caproic acid N-hydroxysuccinimide ester, iodoaceticacid N-hydroxysuccinimide ester, 3-malemidoacetic acidN-hydroxysuccinimide ester, 3-malemidobenzoic acid N-hydroxysuccinimideester, 4-(N-malemido)benzophenone, gamma-malemidobutyric acidN-hydroxysuccinimide ester, epsilon-malemidocaproic acidN-hydroxysuccinimide ester, 4-(N-malemidomethyl)cyclohexanecarboxylicacid N-hydroxysuccinimide ester,4-(N-malemidomethyl)cyclohexanecarboxylic acid3-sulfo-N-hydroxysuccinimide ester, beta-malemidopropionic acidN-hydroxysuccinimide ester,N,N′-bis(3-malemidopropionyl)-2-hydroxy-1,3-propanediamine,1,4-phenylene diisothiocyanate, N,N′-o-phenylene dimalemide,N,N′-p-phenylene dimalemide, polyoxyethylene bis(glycidyl ether),bis(polyoxyethylene bis(glycidyl ether)), polyoxyethylenebis(imidazolylcarbonyl), bis(polyoxyethylene bis(imidazolylcarbonyl)),polyoxyethylene bis(p-nitrophenyl carbonate),3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester, subericacid bis(N-hydroxysuccinimide) ester, succinic acid malemidoethylN-hydroxysuccinimide ester, 1,5 bis(succinimidooxycarbonyloxy)-pentane,and bis(N-succinimidyl) carbonate.

[0129] Ligands of a Cell Surface Protein

[0130] The multifunctional molecules of the present invention compriseone part which is a lectin and is capable of binding to at least onecarbohydrate molecule on an antigen bearing target, and a second partcomprising a ligand for a cell surface protein of an antigen presentingcell. The ligand can be any ligand which binds to one or more of thecell surface molecules indicated by GenBank Accession number in AppendixI or II. More preferably, however, the ligand includes, but is notlimited to an opsonin, a cytokine, a heat shock protein, an adhesionmolecule. a defensin, or a counterreceptor for a T cell costimulatorymolecule; or a portion of any of these molecules, e.g., about (or atleast about) 5, 8, 10, 12, 15, 20, 25, 35, 40, 50, 60, 70, 80, 100, or120 contiguous amino acid residues, up to the full length of such amolecule.

[0131] Cytokines Useful According to the Invention

[0132] The term “cytokine” as defined hereinabove refers to apolypeptide molecule that is naturally secreted by mammalian cells andthat binds to a cell surface receptor on a leukocyte. The term“cytokine” also refers herein to a polypeptide molecule that is a ligandfor a receptor for a naturally occurring cytokine. Unlike an opsonin, acytokine does not naturally contemporaneously bind an antigen and acell-surface receptor.

[0133] Leukocytes which bear receptors for cytokines include, forexample, monocytes, macrophages, dendritic cells, neutrophils,eosinophils, basophils, platelets, lymphocytes, T lymphocytes, Blymphocytes, NK cells, myeloma cells, lymphoma cells, and leukemiccells.

[0134] Without being bound by any one mechanism, it is believed thatcell-surface associated cytokines provide an advantage over freelydiffusible cytokines by allowing stable juxtaposition of the cytokine tothe cell, thus increasing the concentration of cytokine in the vicinityof the cell.

[0135] Preferred cytokines are non-rodent cytokines, e.g primate, e.g.human cytokines.

[0136] Some cytokines can be regarded as belonging to one or morefamilies of cytokines based on structural and/or functional properties.One such family consists of the interleukins. Interleukins arestructurally diverse, but share the property of both being expressed byand acting on leukocytes. Examples of interleukins include IL-1 (e.g.polypeptides encoded by Genbank Accession No. M15330, M28983, E04743,M15131) IL-2 (e.g. polypeptides encoded by Genbank Accession No. E01108,K02797), IL-3 (e.g. polypeptides encoded by Genbank Accession No.A02046, M14743), IL-4 (e.g. polypeptides encoded by M13982, M25892),IL-5 (e.g. polypeptides encoded by X06270, J03478), IL-6 (e.g.polypeptides encoded by E02772, M20572), IL-7 (e.g. polypeptides encodedby J04156, M29054-29057), IL-8 (e.g. polypeptides encoded by M28130),IL-9 (e.g. sequences disclosed in Kelleher et al, Blood. 1991; 77:1436-1441, Immunogenetics 1990;31(4):265-270), IL-10 (e.g. polypeptidesencoded by M84340, U16720), IL-11 (e.g. sequences disclosed in Paul etal, Proc Natl Acad Sci U S A. 1990; 87: 7512-7516, Morris et al, ExpHematol. 1996; 24: 1369-1376), IL-12 (e.g. polypeptides encoded byGenbank Accession No. M86671, S82412; Genbank protein P29459, P29460),IL-13 (e.g. polypeptides encoded by U31120, L13028), IL-14 (e.g.sequences disclosed in Ambrus et al, Proceedings of the National Academyof Science (USA) 1993; 90: 6330-4), IL-15 (e.g. polypeptides encoded byAF031167, U22339), IL-16 (e.g. polypeptides encoded by AF006001,M90391), IL-17 (e.g. polypeptides encoded by U32659, U43088), IL-18(e.g. polypeptides encoded by D49949, D49950), IL-19 (e.g. polypeptidesencoded by AY040367), IL-20 (e.g. polypeptides encoded by NM02130,NM018724), IL-21 (e.g. polypeptides encoded by AF254069, AF254070),IL-22 (e.g. polypeptides encoded by AF279437), IL-23 (e.g. polypeptidesencoded by AF301619, AF301620, AY055379 [p19 alpha chain combines withIL-12 p40 chain to form IL-23]), IL-24 (e.g. polypeptides encoded byAF276916, NM053095), IL-25 (e.g. polypeptides encoded by NM080837),TNF-alpha (e.g. polypeptides encoded by M16441, Y00467), and GM-CSF(e.g. polypeptides encoded by X03019, M11220) and their homologues amongspecies. Nucleotide sequences encoding homologues will hybridize to eachother under moderate- to high-stringency conditions.

[0137] Another family consists of the hematopoietins. Members of thisfamily comprise helical regions, known as helices A, B, C, and D.Helices A and B and helices C and D run roughly parallel to each other,respectively. Examples of hematopoietins include IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, GM-CSF, G-CSF (e.g.polypeptides encoded by Genbank Accession No. E01219, M13926),oncostatin M (e.g. polypeptides encoded by Genbank Accession No. D31942,sequences disclosed in Malik et al, Mol Cell Biol 1989, 9:2847-2853),LIF (e.g. polypeptides encoded by Genbank Accession No. X13967, X06381),CNTF (e.g. polypeptides encoded by Genbank Accession No. U05342,X60542), and their homologues among species. Nucleotide sequencesencoding homologues will hybridize to each other under moderate- tohigh-stringency conditions.

[0138] Human IL2 is a protein of 133 amino acids (15.4 kDa) with aslightly basic pI. Murine and human IL2 display a homology ofapproximately 65%. IL2 is synthesized as a precursor protein of 153amino acids with the first 20 amino-terminal amino acids functioning asa hydrophobic secretory signal sequence. The protein contains a singledisulfide bond (positions Cys58/105) essential for biological activity.

[0139] IL2 is O-glycosylated at threonine at position 3. Variants withdifferent molecular masses and charges are due to variableglycosylation. Non-glycosylated IL2 is also biologically active.Glycosylation appears to promote elimination of the factor byhepatocytes.

[0140] A dimeric form of human IL2, produced by the action of atransglutaminase isolated from regenerating fish optic nerves, has beenshown to be a cytotoxic factor for rat brain oligodendrocytes inculture.

[0141] The human IL2 gene contains four exons. The IL2 gene maps tohuman chromosome 4q26-28 (murine chromosome 3). The homology of murineand human IL2 is 72% at the nucleotide level in the coding region.

[0142] The biological activities of IL2 are mediated by a membranereceptor that is expressed almost exclusively on activated, but not onresting, T-cells at densities of 4-12×10³ receptors/cell. ActivatedB-cells and resting mononuclear leukocytes rarely express this receptor.The expression of the IL2 receptor is modulated by IL5 and IL6. Threedifferent types of IL2 receptors are distinguished that are expresseddifferentially and independently. The high affinity IL2 receptor (Kdis˜10 pM) constitutes approximately 10% of all IL2 receptors expressed bya cells. This receptor is a membrane receptor complex consisting of thetwo subunits IL2R-alpha (TAC antigen=T-cell activation antigen; p55) andIL2R-beta (p75; CD 122) as the ligand binding domains and a gamma chainas a signaling component. p75 is expressed constitutively on restingT-lymphocytes, NK-cells, and a number of other cell types while theexpression of p55 is usually observed only after cell activation. p55is, however, synthesized constitutively by a number of tumor cells andby HTLV-1-infected cells.

[0143] IL2 receptor expression of monocytes is induced by IFN-gamma, sothat these cells become tumor-cytotoxic. In T-cells the expression ofp75 can be reduced by IL3. An intermediate affinity IL2 receptor(Kdis=100 pM) consists of the p75 subunit and a gamma chain (see below)while a low affinity receptor (Kdis=10 nM) is formed by p55 alone.

[0144] p55 (e.g. polypeptides encoded by Genbank Accession No. X01057)has a length of 251 amino acids with an extracellular domain of 219amino acids an a very short cytoplasmic domain of 13 amino acids. Thep55 gene maps to human chromosome 10p14-p15.

[0145] p75 (e.g. polypeptides encoded by Genbank Accession No. M26062,M28052) has a length of 525 amino acids with an extracellular domain of214 amino acids and a cytoplasmic domain of 286 amino acids. The p75gene contains 10 exons and has a length of approximately 24 kb. It mapsto human chromosome 22q11.2-q12 and to murine chromosome 15 (band E).

[0146] A third 64 kDa subunit of the IL2 receptor, designated gamma, hasbeen described (e.g. polypeptides encoded by Genbank Accession No.D13821, D11086). Murine and human gamma subunits of the receptor haveapproximately 70% sequence identity at the nucleotide and amino acidlevels. This subunit is required for the generation of high andintermediate affinity IL2 receptors but does not bind IL2 by itself.These two receptor types consist of an alpha-beta-gamma heterotrimer anda beta-gamma heterodimer, respectively. The gene encoding the gammasubunit of the IL2 receptor maps to human chromosome Xq13, spansapproximately 4.2 kb and contains eight exons. The gamma subunit of theIL2 receptor has been shown recently to be a component of the receptorsfor IL4 and IL7. It is also believed to be a component of the IL13receptor.

[0147] The amino acids at positions 267-317 lying directly adjacent tothe transmembrane region of p75 are involved in IL2-mediated signaltransduction. In addition the IL2 receptor is associated with a numberof other proteins (p22, p40, p100) which are thought to be involved inmediating conformational changes in the receptor chains,receptor-mediated endocytosis, and further signal transductionprocesses. One of the identified proteins is the 95 kDa cell adhesionmolecule ICAM-1 which probably focuses IL2 receptors at regions ofcell-to-cell contacts and thus may mediate paracrine activities, forexample, during IL2-mediated stimulation of T-cells. Another proteinassociated with p75 is a tyrosine-specific protein kinase called lck.The observation that proliferation of cells induced by IL2 is inhibitedby specific inhibitors of protein tyrosine kinases in an lck negativecell line suggests that other kinases may also be associated with IL2receptors. Two such kinases, called fyn and lyn, have been identified.In addition, IL2 receptor signaling may also be mediated by vav.

[0148] Activated lymphocytes continuously secrete a 42 kDa fragment ofthe TAC antigen. This fragment circulates in the serum and plasma andfunctions as a soluble IL2 receptor ( sIL2R ). The concentrations ofthis soluble receptor vary markedly in different pathologicalsituations, for example, infections, autoimmune diseases, leukemias, orafter organ transplantation. Levels may increase up to 100-fold. Thelevels of sIL2R appear to correlate with the severity of HIV-inducesdiseases and may be of diagnostic value also in other settings.

[0149] Mouse and human IL2 both cause proliferation of T-cells of thehomologous species at high efficiency. Human IL2 also stimulatesproliferation of mouse T-cells at similar concentrations, whereas mouseIL2 stimulates human T-cells at a lower (sixfold to 170-fold)efficiency.

[0150] IL2 is a growth factor for all subpopulations of T-lymphocytes.It is an antigen-unspecific proliferation factor for T-cells thatinduces cell cycle progression in resting cells and thus allows clonalexpansion of activated T-lymphocytes. This effect is modulated byhormones such as prolactin.

[0151] IL2 also promotes the proliferation of activated B-cells alsothis requires the presence of additional factors, for example, IL4.

[0152] Due to its effects on T-cells and B-cells IL2 is a centralregulator of immune responses. It also plays a role in anti-inflammatoryreactions, in hematopoiesis and in tumor surveillance. IL2 stimulatesthe synthesis of IFN-gamma in peripheral leukocytes and also induces thesecretion of IL1, TNF-alpha and TNF-beta.

[0153] It is believed that he induction of the secretion of tumoricidalcytokines apart from the activity in the expansion of LAK cells(lymphokine-activated killer cells ) are probably the main factorsresponsible for the antitumor activity of IL2.

[0154] IL2 can be assayed in bioassays employing cell lines that respondto the factor (e.g., ATH8, CT6, CTLL-2, FDCPmix, HT-2, NKC3, TALL-103 ).Specific ELISA assays for IL2 and enzyme immunoassays for the solublereceptor are also available. The soluble receptor can be detected alsoby employing biotinylated IL2 and flow-through cytometry or ELISAassays.

[0155] IL2 displays significant anti-tumor activity for a variety oftumor cell types since it supports the proliferation and clonalexpansion of T-cells that specifically attack certain tumors. IL2 isincreasingly used to treat patients with cancers refractory toconventional treatment. Combination therapy with systemicallyadministered IL2 has resulted in long-term remissions in 30% of patientswith metastatic renal cell carcinoma, for which there is no standardtreatment. Objective and long-lived clinical responses have beendocumented also in a proportion of patients with melanoma or acutemyeloid leukemia.

[0156] High dose systemic IL2 therapy is also associated with a greatnumber of unwanted toxic side-effects. IL2 has additional effects onother components of the cellular immune system, including B-cells andmacrophages, and induces the secretion of other soluble mediators,including TNF-alpha, TNF-beta, and IFN-gamma. These effects maycontribute to the antitumor activity of IL2 as well as to itsdose-related toxicity.

[0157] The transduction of murine tumor cells with a functional IL2 genehas been shown to lead to the rejection of the genetically modifiedcells by syngeneic hosts. Altered tumor cells expressing IL2 alsoincrease systemic immunity.

[0158] Human IL4 is a protein of 129 amino acids (20 kDa) that issynthesized as a precursor containing a hydrophobic secretory signalsequence of 24 amino acids. IL4 is glycosylated at two arginine residues(positions 38 and 105) and contains six cysteine residues involved indisulfide bond formation. The disulfide bonds are essential forbiological activity. Some glycosylation variants of IL4 have beendescribed that differ in their biological activities. A comparison ofmurine and human IL4 shows that both proteins only diverge at positions91-128.

[0159] An IL4 variant, Y124D, in which Tyr124 of the recombinant humanprotein is substituted by an aspartic acid residue, binds with highaffinity to the IL4 receptor (Kd=310 pM). This variant is a powerfulantagonist for the IL4 receptor system. It retains no detectableproliferative activity for T-cells and competitively inhibitsIL4-dependent T-cell proliferation (K(i)=620 pM). The existence of thismutant demonstrates that high affinity binding and signal generation canbe uncoupled efficiently in a ligand. Y124D also acts as a powerfulantagonist for the IL13 receptor.

[0160] The human IL4 gene contains four exons and has a length ofapproximately 10 kb. It maps to chromosome 5q23-31. The murine gene mapsto chromosome 11. The IL4 gene is in close proximity to other genesencoding hematopoietic growth factors (e.g., GM-CSF, M-CSF, IL3, IL5).The distance between the IL4 and the IL5 gene is approximately 90-240kb.

[0161] At the nucleotide level the human and the murine IL4 gene displayapproximately 70% homology. The 5′ region of the IL4 contains severalsequence elements, designated CLE (conserved lymphokine element), thatare binding sites for transcription factors controlling the expressionof this and other genes. A sequence motif, called P sequence(CGAAAATTTCC; SEQ ID NO: 1) in the 5′ region of the human IL4 gene(positions-79--69) is the binding site for a nuclear factor, calledNF(P), mediating the response to T-cell activation signals.

[0162] The biological activities of IL4 are mediated by a specificreceptor (Kdis=20-100 pM) which is expressed at densities of 100-5000copies/cell (e.g. polypeptides encoded by Genbank Accession No. M29854,X52425). The extracellular domain of the IL4 receptor is related to thereceptors for erythropoietin (Epo), IL6, and the beta chain of the IL2receptor. It has been given the name CD124.

[0163] The cDNA for the murine IL4 receptor encodes a transmembraneprotein of 810 amino acids (including a secretory signal sequence). Thisreceptor has a large intracellular domain of 553 amino acids. The humanreceptor has an extracellular domain of 207 amino acids, a transmembranedomain of 24 residues, and a large intracellular domain of 569 aminoacids.

[0164] The IL4 receptor has been shown recently to contain the gammasubunit of the IL2 receptor as a signaling component. This gamma subunitis also associated with the receptors for IL4 and IL7 and probably alsoof IL13. Two forms of the receptor have been described, one of which issecreted. The secreted receptor only contains the extracellular IL4binding domain and is capable of blocking IL4 activities. An IL4 bindingprotein (IL4-BP) that binds IL4 with the same affinity as the IL4receptor has been shown also to be a soluble IL4 receptor variant. Thesesoluble receptors probably function as physiological regulators ofcytokine activities by inhibiting receptor binding or act as transportproteins. Soluble receptors or binding proteins have been described alsofor IL1 (IL1 receptor antagonist), IL2, IL6, IL7, TNF-alpha, IGF, andIFN-gamma.

[0165] The biological activities of IL4 are species-specific; mouse IL4is inactive on human cells and human IL4 is inactive on murine cells.IL4 promotes the proliferation and differentiation of activated B-cells,the expression of class II MHC antigens, and of low-affinity IgEreceptors in resting B-cells. IL4 enhances expression of class II MHCantigens on B-cells. It can promote their capacity to respond to otherB-cell stimuli and to present antigens for T-cells. This may be one wayto promote the clonal expansion of specific B-cells and the immunesystem may thus be able to respond to very low concentrations ofantigens. The production of IL4 by non-B non-T-cells is stimulated ifthese cells interact with other cells via their Fc receptors for IgE orIgG. This effect can be enhanced by IL3. IL2 and PAF (plateletactivating factor) induce the synthesis of IL4 while TGF-beta inhibitsit.

[0166] IL3 antagonizes the IL2-induced effects in B-cells and causes aslow decrease of the expression of IL2 receptors, thus inhibiting theproliferation of human B-cells stimulated by IL2. In activated B-cellsIL4 stimulates the synthesis of IgG1 and IgE and inhibits the synthesisof IgM, IgG3, IgG2a and IgG2b. This isotype switching induced by IL4 inB-cells is antagonized by IFN-gamma. The growth of multiple myelomas canbe suppressed by IL4 which inhibits the synthesis of IL6, a myelomagrowth factor. IL4 also inhibits the synthesis of IL6 in human alveolarmacrophages.

[0167] Pretreatment of macrophages with IL4 prevents the production ofIL1, TNF-alpha and prostaglandins in response to activation of the cellsby bacterial endotoxins or IFN-gamma.

[0168] IL4 synergises with Epo and G-CSF/Epo in the generation ofcolonies containing granulocytes or erythroid progenitor cells in acolony formation assay.

[0169] The classical detection method for IL4 is a B-cell costimulationassay measuring the enhanced proliferation of stimulated purifiedB-cells. IL4 can be detected also in bioassays, employing IL4-responsivecells (e.g., BALM-4; BCL1; CT.4S; CTL44; CTLL-2; Da; FDCPmix ; HT-2; L4;L138.8A; MO7E; MC/9; NFS-60; Ramos, Sez627, TF-1; TS1). A specificdetection method for human IL4 is the induction of CD23 in a number ofB-cell lines with CD23 detected either by flow-through cytometry or by afluorescence immunoassay. An immunoassay that allows rapid determinationof the rate of IL4 production under conditions preventingconsumption/degradation is cytokine immunotrapping.

[0170] IL4 inhibits the growth of colon and mammary carcinomas. It hasbeen shown to augment the development of LAK cells. The transduction ofmurine tumor cells with a functional IL4 gene has been shown to lead tothe rejection of the genetically modified cells by syngeneic hosts.Altered tumor cells expressing IL4 also increase systemic immunity. Micevaccinated with transduced cells reject a subsequent challenge ofnon-transduced cells, and, in some cases, a pre-existing tumor.

[0171] Human IL6 is a protein of 185 amino acids glycosylated atpositions 73 and 172. It is synthesized as a precursor protein of 212amino acids. Monocytes express at least five different molecular formsof IL6 with molecular masses of 21.5-28 kDa. They mainly differ bypost-translational alterations such as glycosylation andphosphorylation.

[0172] IL6 isolated from various cell types shows somemicroheterogeneity in its N terminus. A 42-45 kDa form has been observedin plasma that is probably complexed with a carrier protein,alpha-2-macroglobulin (α2M). Murine and human IL6 show 65% sequencehomology at the DNA level and 42% homology at the protein level.

[0173] IL6 is a member of a family of cytokines which also includes LIF,CNTF, Oncostatin M, IL11, and CT-1. All known members of the IL6cytokine family induce hepatic expression of acute phase proteins.

[0174] A stable and highly bioactive designer cytokine consisting of afusion protein between IL6 and a soluble IL6 receptor, designated H-IL6,has been used for human hematopoietic progenitor cell expansion and isuseful in cases in which cells do not respond to IL6 but require astable complex consisting of IL6 and a soluble IL6 receptor.

[0175] The human IL6 gene has a length of approximately 5 kb andcontains five exons. It maps to human chromosome 7p21-p14 between themarkers D7S135 and D7S370. The murine gene maps to chromosome 5. Thenucleotide sequences of IL6 and G-CSF genes resemble each other in a waysuggesting a possible evolutionary relationship.

[0176] The IL6 receptor (e.g. polypeptides encoded by Genbank AccessionNo. M20566, E03515) is expressed on T-cells, mitogen-activated B-cells,peripheral monocytes and some macrophage- and B-cell-derived tumor celltypes. It is not expressed in resting B-cells but is in resting T-cells.In hepatocytes the IL6 receptor expression is enhanced after treatmentwith IL6 or IL1. In several cell types the expression of the IL6receptor is also enhanced by glucocorticoids. The IL6 receptor gene mapsto human chromosome 1q21.

[0177] The IL6 receptor is a strongly glycosylated protein of 80 kDa anda length of 449 amino acids. It has been designated CD126. It issynthesized as a precursor of 468 amino acids. The molecular structureresembles that of receptors for M-CSF, PDGF and IL1 in that the receptorcontains an immunoglobulin-like sequence domain in the aminoterminalregion of the extracellular receptor domain.

[0178] The intracellular domain of the IL6 receptor has a length ofapproximately 82 amino acids and does not show any homology to otherproteins involved in intracellular signal transduction. Two differentforms of the receptor have been described that bind IL6 with differentaffinities (Kdis=10⁻⁹ and 10⁻¹¹ M) and most likely arise bypost-translational modification of the same receptor protein. Biologicalactivities of IL6 have been found also at concentrations of 10⁻¹³-10⁻¹⁵M suggesting either the existence of other high-affinity receptorconformations or the existence of further receptor molecules with higheraffinities.

[0179] IL6 receptor-mediated signal transduction involves protein kinaseC and also adenylate cyclase.

[0180] The complex formed between IL6 and its receptor associates with atransmembrane glycoprotein, gp130 (918 amino acids; cytoplasmic domainof 277 amino acids), that is involved in signal transduction. Binding ofIL6 to its receptor leads to disulfide-linked homodimerization of gp130and the associated activation of a tyrosine kinase as the first step ofsignal transduction. gp130 is expressed also in cells that do notexpress IL6 receptors. It has been found to be a component of otherreceptors, including those for IL11, LIF, Oncostatin M, and CNTF, andCT-1. This explains why LIF, CNTF, and IL6 share many biologicalactivities although the factors themselves are not related to eachother. A factor resembling STAT proteins, termed LIL factor, has beenfound to be involved in signaling pathways of IL6, and also of IL1 andbacterial lipopolysaccharides.

[0181] A soluble form of the IL6 receptor (IL6R-SUP (IL6 receptorsoluble urinary protein)) has been described also that also interactswith gp130. These soluble receptors probably function as physiologicalregulators of cytokine activities by inhibiting receptor binding or actas transport proteins. Similar soluble receptors or binding proteinshave been described also for IL1 ( IL1ra, IL1 receptor antagonist), IL2,IL4, IL7, TNF-alpha, IGF, and IFN-gamma.

[0182] Some cells, including hematopoietic progenitor cells and neuronalcells, are only responsive towards a combination of IL6 and soluble IL6receptor but not to IL6 alone.

[0183] Human IL6 is biologically active in monkeys, rats, and mice.Murine IL6 is not active in human cells. The plethora of biologicalactivities is exemplified by the many different acronyms under which IL6has been described. IL6 is a pleiotropic cytokine influencingantigen-specific immune responses and inflammatory reactions. It is oneof the major physiological mediators of cute phase reaction. Inhepatocytes IL6 in combination with glucocorticoids induces thesynthesis of metallothioneins and increases intracellular zinc levels,thus preventing CCL4-induced hepatotoxicity. IL6 is a neurotrophicfactor for cholinergic neurons that promotes their survival in culture.Some neuronal cell lines can be induced to differentiate by IL6.

[0184] IL6, like IL1, stimulates the synthesis of ACTH (Corticotropin)in the pituitary. Glucocorticoids synthesized in response to ACTHinhibit the production of IL6, IL1 and TNF in vivo, thus establishing asort of negative feedback loop between the immune system andneuroendocrine functions. In astrocytes IL6 induces the synthesis ofNerve Growth Factor (NGF).

[0185] IL6 is a B-cell differentiation factor in vivo and in vitro andan activation factor for T-cells. In the presence of IL2 IL6 induces thedifferentiation of mature and immature T-cells into cytotoxic T-cells.IL6 also induces the proliferation of thymocytes and probably plays arole in the development of thymic T-cells.

[0186] IL6 is capable of inducing the final maturation of B-cells intoimmunoglobulin-secreting plasma cells if the cells have beenpre-activated by IL4. In B-cells IL6 stimulates the secretion ofantibodies to such a degree that serum IgG1 levels can rise120-400-fold.

[0187] IL6 at concentrations of only 0.002 ng/mL is one of the majorautocrine growth modulator for many human myelomas. The growth of thesecells can be inhibited by monoclonal antibodies directed against IL6. Itcan be inhibited also by the introduction of antisense oligonucleotidesagainst IL6 or by IL4. The growth-inhibitory effects of corticosteroidson myeloma cells is probably due to the steroid-induced reduction in theexpression of IL6. The growth of human IL6 dependent myeloma cells canbe inhibited also by IFN-gamma. IL6 may also function as an autocrinegrowth modulator for other tumor types, some of which have been found tosecrete IL6 constitutively. IL6 has been shown to be an autocrinemodulator of growth for in vitro cervical tumor cell growth. On theother hand IL6 blocks the growth of some solid tumors such as mammarycarcinomas, cervical carcinomas, human lung cancer cell lines,histiocytic lymphomas, and melanomas.

[0188] IL6 and IL3 synergise in vitro in promoting the proliferation ofmultipotent hematopoietic progenitor cells. IL6 is also a thrombopoietinthat induces the maturation of megakaryocytes in vitro and increasesplatelet counts in vivo. In murine, but not in human bone marrowcultures IL6 shows activities resembling those of GM-CSF.

[0189] Plasmacytoma cells produce IL6 and also the IL6 receptor. It hasbeen suggested that these cells are stimulated in an autocrine fashion.A paracrine mechanism involving the presence of two different cellpopulations, one producing the factor and the other expressing thereceptor, has been described also.

[0190] IL6 can be detected in bioassays employing IL6 responsive celllines (e.g., 7TD1 ; B9; CESS, KPMM2, KT-3; M1, MH60-BSF-2, MO7E; MonoMac 6; NFS-60; PIL-6; SKW6-C14; T1165; XG-1). IL6 can be assayed also byits activity as a hybridoma growth factor. Sensitive immunoassays andcolorimetric tests are also available. An ELISA assay exists fordetecting the receptor-associated gp130 protein.

[0191] In combination with other cytokines (for example, IL2) IL6 may beuseful in the treatment of some tumor types. The transduction of murinetumor cells with a functional IL6 gene has been shown to lead to therejection of the genetically modified cells by syngeneic hosts. Alteredtumor cells expressing IL6 also increase systemic immunity. Micevaccinated with transduced cells reject a subsequent challenge ofnon-transduced cells, and, in some cases, a pre-existing tumor.

[0192] Human IL10 is a homodimeric protein with subunits having a lengthof 160 amino acids. Human IL10 shows 73% amino acid homology with murineIL10. The human IL10 contains four exons. It is closely related to theproduct of the BCRF-1 gene (Bam HI C fragment rightward reading frame)of Epstein-Barr virus (84% homology at the protein level). These twoproteins are more closely related to each other than human and murineIL10. BCRF-1 has therefore also been called viral IL10 (vIL10). Thehuman IL10 gene maps to chromosome 1. The human IL10 shows 81% homologywith murine IL10 at the nucleotide level.

[0193] A receptor has been identified on murine and human cells by usingradiolabeled IL10 (e.g. polypeptides encoded by Genbank Accession No.L12120, U00672). Mouse IL10 is capable of blocking binding of human IL10to mouse but not human cells. The murine IL10 receptor has been cloned.This receptor is a protein of approximately 110 kDa that binds murineIL10 specifically. This receptor is structurally related to receptorsfor IFN.

[0194] IL10 inhibits the synthesis of a number of cytokines such asIFN-gamma, IL2 and TNF-beta in Th1 subpopulations of T-cells but not ofTh2 cells. This activity is antagonized by IL4. The inhibitory effect onIFN-gamma production is indirect and appears to be the result of asuppression of IL12 synthesis by accessory cells. In the human system,IL10 is produced by, and down-regulates the function of, Th1 and Th2cells. In macrophages stimulated by bacterial lipopolysaccharides IL10inhibits the synthesis of IL1, IL6 and TNF-alpha by promoting, amongother things, the degradation of cytokine mRNA. It also leads to aninhibition of antigen presentation. In human monocytes IFN-gamma andIL10 antagonize each other's production and function. IL10 has beenshown also to be a physiologic antagonist of IL12.

[0195] IL10 also inhibits mitogen- or anti-CD3-induced proliferation ofT-cells in the presence of accessory cells and reduces the production ofIFN-gamma and IL2. Exogenous IL2 and IL4 inhibit theproliferation-inhibitory effect but do not influence the production ofIFN-gamma. In LPS-stimulated macrophages IFN-gamma increases thesynthesis of IL6 by inhibiting the production of IL10. IL10 appears tobe responsible for most or all of the ability of Th2 supernatants toinhibit cytokine synthesis by Th1 cells.

[0196] IL10 inhibits secretion of Ig by T-cell-independent antigensinduced by IL5 but not that induced by IL2.

[0197] Murine Ly-1 B cells are the principal source of IL10. In contrastto other B-cells, Ly-1 B-cells express greatly elevated constitutive andinducible levels of IL10. These cells also have the distinctive propertyof continuous self-replenishment. The continuous treatment of newbornmice with anti-IL10 antibodies leads to a depletion of the Ly-1 B-cellswhile maintaining a normal population of splenic B-cells. These micealso contain greatly reduced serum immunoglobulin M levels and are alsoimpaired in their antibody responses to specific antigens. IL10 istherefore a regulator of Ly-1 B-cell development. The mechanism of Ly-1B-cell depletion appears to involve the increased production ofIFN-gamma since co-administration of neutralizing anti-IFN-gammaantibodies substantially restores the number of peritoneal-resident Ly-1B-cells in these mice.

[0198] IL10 is also a costimulator for the growth of mature and immaturethymocytes (together with IL2, IL4 and IL7) and functions as a cytotoxicT-cell differentiation factor, promoting a higher number ofIL2-activated cytotoxic T-lymphocyte precursors to proliferate anddifferentiate into cytotoxic effector cells. IL10 sustains viability ofB-cells in vitro and also stimulates B-cells and promotes theirdifferentiation. It enhances the expression of MHC class II antigens onB-cells whereas it inhibits MHC class II expression on monocytes. InB-cells activated via their antigen receptors or via CD40 IL10 inducesthe secretion of IgG, IgA and IgM. This effect is synergised by IL4while the synthesis of immunoglobulins induced by IL10 is antagonized byTGF-beta. The activation of macrophages can be prevented by IL10.

[0199] It has been shown that human IL10 is a potent and specificchemoattractant for human T-lymphocytes. The chemotactic activity isdirected towards cells expressing CD8 and not towards CD4 (+)cells. IL10also inhibits the chemotactic response of CD4 (+)cells, but not of CD8(+)cells, towards IL8. IL10 can be detected with a sensitive ELISAassay. The murine mast cell line D36 can be used to bioassay human IL10.The intracellular factor can be detected also by flow cytometry.

[0200] The introduction of an IL10 expression vector into CHO cells hasbeen used to analyze the consequences of local IL10 production in vivo.These altered cells were no longer tumorigenic in nude mice or severecombined immunodeficient SCID mice and also suppressed the growth ofequal numbers of co-injected normal CHO cells. While normal CHO tumorsare usually substantially infiltrated by macrophages, these werevirtually absent within CHO-IL10 tumor tissues, suggesting that IL10indirectly suppresses tumor growth of certain tumors by inhibitinginfiltration of macrophages which may provide tumor growth promotingactivity.

[0201] Human IL12 is a heterodimeric 70 kDa glycoprotein consisting of a40 kDa subunit (p40, 306 amino acids; 10% carbohydrate) and a 35 kDasubunit (p35, 197 amino acids; 20% carbohydrate) linked by disulfidebonds that are essential for the biological activity of IL12. p40contains 10 cysteines and a binding site for heparin; p35 contains 7cysteines.

[0202] The two subunits of IL12 are not related to any other knownproteins. p40 shows some homology with the extracellular domain of thereceptor for IL6, and p35 appears to be a homologue of IL6.

[0203] Bioactive murine and human IL12 fusion proteins combining the twoIL12 subunits in a single molecule have been described. This designercytokine retains antitumor activity in vivo. Flexi 12, a single chainprotein retaining all of the biological characteristics of the dimericrecombinant IL12, has also been described.

[0204] The gene encoding the p40 subunit of IL12 (IL12B) maps to humanchromosome 5q31-q33 in the same region that also harbors other cytokinegenes. The gene encoding the p35 subunit of IL12 (IL12A) maps to humanchromosome 3p12-q13.2. The expression of the two genes is regulatedindependently of each other.

[0205] The IL12 receptor appears to be a single protein of approximately110 kDa (e.g. polypeptides encoded by Genbank Accession No. U03187,U23922, U64198, U64199). Up to 1000-9000 high affinity IL12receptors/cell are expressed on peripheral blood mononuclear cellsactivated by various T-cell mitogens or by IL2. IL12 receptors arepresent on activated T-cells expressing CD4 and CD8 and on activatedCD56 positive natural killer cells. Resting peripheral blood mononuclearcells, tonsillar B-cells, or tonsillar B-cells activated by anti-IgM/Dx,anti-IgM/Dx+IL2, or SAC+IL2 do not express the receptor. High affinityIL12 receptors are expressed constitutively on a transformed marmosetNK-like cell line, HVS.SILVA 40.

[0206] Binding of IL12 to its receptor can be prevented by monoclonalantibodies directed against the p40 subunit which therefore contains thebinding site. The p40 subunit of IL12 shows homology with theextracellular domain of the IL6 receptor. A virus-encoded homologue ofthe p40 subunit is EBV-induced gene-3.

[0207] Human IL12 is not active in murine lymphocytes. Hybridheterodimers consisting of murine p35 and human p40 subunits retainbioactivity on murine cells; however, the combination of human p35 andmurine p40 is completely inactive on murine cells. Murine IL12 is activeon both murine and human lymphocytes. The p40 subunit of murine IL12subunit p40 (IL12p40) has been shown to specifically antagonize theeffects of the IL12 heterodimer in different assay systems and tofunction as an endogenous specific inhibitor for the IL12 heterodimer.

[0208] IL12 stimulates the proliferation of human lymphoblasts activatedby phytohemagglutinin. IL12 activates NK-cells positive for CD56, andthis activity is blocked by antibodies specific for TNF-alpha. IL12promotes specific allogenic CTL reactions. IL12 synergizes also withanti-CD3 antibodies and with allogeneic stimulation in mixed lymphocytecultures in inducing T-cell proliferation.

[0209] In peripheral lymphocytes of the Th1 type IL12 induces thesynthesis of IFN-gamma and IL2, and TNF. TNF-alpha also appears to beinvolved in mediating the effects of IL12 on natural killer cells sincethe effects of IL12 are inhibited by an antibody directed againstTNF-alpha. IL12 and TNF-alpha are costimulators for IFN-gamma productionwith IL12 maximizing the IFN-gamma response; the production of IL12,TNF, and IFN-gamma is inhibited by IL10. In Th2 helper cells IL12reduces the synthesis of IL4, IL5, and IL10.

[0210] IL12 synergises with suboptimal amounts of IL2 in promoting theproliferation of mononuclear cells in the peripheral blood and inpromoting the generation of LAK cells (lymphokine activated killercells). Picomolar concentrations of IL12 are as effective as nanomolarconcentrations of IL2 in augmenting the cytolytic activity of naturalkiller cells expanded in vivo by IL2. IL12 also acts as a co-mitogen andpotentiates the proliferation of resting peripheral cells induced byIL2.

[0211] IL12 enhances myelopoiesis of primitive bone marrow progenitorcells induced by SCF (stem cell factor) and synergizes with colonystimulating factors to induce proliferation. IL12 also has synergisticeffects on more committed bone marrow progenitors, synergising with IL3,IL11, or IL3 plus SCF.

[0212] IL12 is of potential clinical interest since it allows thereduction of doses of IL2 required for the generation of LAK cells(lymphokine-activated killer cells). IL12 has been shown to inhibit thegrowth of a variety of experimental tumors in vivo and to haveantiangiogenic effects in vivo, which are, at least in part, mediated byIFN-gamma. IL12 therefore seems to be a potential candidate also for thetreatment of angiogenesis-dependent malignancies.

[0213] IL19 and IL10 share 21 percent amino acid identity and areprobably homologs. In monocytes treatment with bacteriallipopolysaccharides induces the synthesis of IL19 and this effect ispotentiated in the presence of IL4 or IL13 but is unaffected byIFN-gamma. GM-CSF directly induces IL19 gene expression in monocytes.IL19 has been shown to bind to the IL20 receptor complex.

[0214] References: Dumoutier L et al Cutting edge: STAT activation byIL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types.Journal of Immunology 167(7): 3545-9 (2001); Gallagher G et al Cloning,expression and initial characterization of interleukin-19 (IL-19), anovel homologue of human interleukin-10 (IL-10). Genes Immun 1(7):442-50 (2000).

[0215] IL20 is structurally related to IL10. IL20 appears to be anautocrine factor for keratinocytes that regulates their participation ininflammation. Overexpression of IL20 in transgenic mice causes neonatallethality with skin abnormalities characterized by an impairment ofepidermal differentiation.

[0216] An IL20 receptor has been identified to consist of two orphanclass 2 cytokine receptor subunits. The receptor is expressed in skinand its expression is upregulated dramatically in psoriatic skin.Engagement of the receptor in a keratinocyte cell line involvessignaling by one member of the STAT proteins, STAT3.

[0217] The IL20 receptor complex has been shown to bind also IL19 andIL24.

[0218] References: Blumberg H et al Interleukin 20: discovery, receptoridentification, and role in epidermal function. Cell 104(1): 9-19(2001); Dumoutier L et al Cutting edge: STAT activation by IL-19, IL-20and mda-7 through IL-20 receptor complexes of two types. Journal ofImmunology 167(7): 3545-9 (2001); Rich B E and Kupper T S Cytokines:IL-20- a new effector in skin inflammation. Current Biology 11(13):R531-4 (2001).

[0219] IL21 has been isolated by Parrish-Novak et al from a cDNA libraryderived from activated CD3 (+)T-cells in a search for the ligand of atype-1 cytokine receptor isolated previously. The cDNA encodes asecreted protein of 131 amino acids protein most closely related to IL2and IL15. The IL21 gene maps to human chromosome 4q26-q27 near the IL2gene. IL21 mRNA is expressed in CD4 (+) but not in CD8 (+) T-cells aftercell activation. It is not expressed also in B-cells and monocytes.

[0220] IL21 stimulates proliferation of B-cell stimulated bycrosslinking of the CD40 antigen. It inhibits proliferation stimulatedby IL4 plus anti-IgM. IL21 augments stimulation of the proliferation ofnaive (CD45RA (+)) but not memory (CD45RO (+)) T-cells mediated byengagement of CD3. IL21 stimulates the proliferation of bone marrowprogenitor cells and the expression of the NK-cell marker CD56 in thepresence of IL15.

[0221] The IL21 receptor has been isolated by Parrish-Novak et al andfound to be expressed by CD23 (+)B-cells, B-cell lines, a T-cellleukemia line, and NK-cell lines. The receptor gene has been mapped tohuman chromosome 16p12. The same receptor has been isolated by Ozaki etal, who called it NILR (novel interleukin receptor). The receptor (538amino acids) is most closely related to human IL2 beta receptor. Thereceptor contains a WSXWS motif in the extracellular region, typical oftype-1 cytokine receptors. The receptor is expressed on NK-cells,T-cells, and B-cell lines.

[0222] The common gamma chain, which is an indispensable subunit of thefunctional receptor complexes for IL2, IL4, IL7, IL9, and IL15 has beenshown also to be part of the IL21 receptor complex. The functionalsignalling complex activates Janus kinases JAK1, JAK3, and the STATproteins STAT1, and STAT3 (Asao et al).

[0223] References: Asao H et al Cutting edge: the common gamma-chain isan indispensable subunit of the IL-21 receptor complex. Journal ofImmunology 167(1):1-5 (2001); Ozaki K et al Cloning of a type I cytokinereceptor most related to the IL-2 receptor beta chain. Proceedings ofthe National Academy of Science (USA) 97: 11439-11444 (2000);Parrish-Novak J et al Interleukin 21 and its receptor are involved in NKcell expansion and regulation of lymphocyte function. Nature 408: 57-63(2000). IL22 (180 amino acids including a signal sequence; 25 kDa; alsocalled IL-TIF) was identified by a cDNA subtraction method as a geneinduced specifically by IL9 in mouse T lymphocytes. The protein showslimited homology with IL10 (22 percent amino acid identity). Human andmurine IL-TIF proteins share 79 percent amino acid identity.

[0224] The murine and human IL-TIF genes both consist of 6 exons. Thehuman single-copy gene maps to chromosome 12q15 (90 Kb from theIFN-gamma gene, and 27 Kb from the AK155 gene encoding anotherIL10-related cytokine. In mice the gene is located also in the sameregion as the IFN-gamma gene. In BALB/c and DBA/2 mice the gene is asingle copy gene. In C57B1/6, FVB and 129 mice the gene is duplicated.The two copies, termed IL-TIF-alpha andIL-TIF-beta show 98 percentnucleotide identity in the coding region and differ by a deletion of 658nucleotide in IL-TIF-beta. This gene may be inactive.

[0225] Expression of IL-TIF is induced by IL9 in thymic lymphomas,T-cells, and mast cells, and by lectins in freshly isolated splenocytes.IL-TIF expression in T-cells does not require protein synthesis, anddepends on the activation Janus kinases and STAT proteins. IL-TIF isexpressed constitutively in thymus and brain.

[0226] In HepG2 human hepatoma cells IL-TIF up-regulates the productionof acute phase proteins. IL-TIF also acts as a pro-inflammatory cytokinein vivo because injection of the protein also induces the synthesis ofacute phase proteins. Synthesis of IL-TIF is induced rapidly afterinjection of bacterial lipopolysaccharides. In contrast to IL10, IL22does not inhibit the production of pro-inflammatory cytokines bymonocytes in response to bacterial lipopolysaccharides. It also does notimpair IL10 function on monocytes. IL-TIF has some inhibitory effects onIL4 production from Th2 T-helper cells.

[0227] IL10 and IL-TIF utilise a common receptor subunit. Antibodiesdirected against the beta chain of the IL10 receptor block the inductionof acute phase proteins by IL-TIF. The functional IL-TIF receptorcomplex consists of two receptor chains. One chain has been identifiedas the orphan receptor CRF2-4 that is expressed in normal liver andkidney. The other chain is the IL10 receptor-2, the second chain of theIL10 receptor complex. Monkey COS expressing CRF2-9 alone respond toIL-TIF. In hamster cells both chains must be expressed to yieldfunctional IL-TIF receptors. Although both receptor chains can bindIL-TIF independently binding of IL-TIF to the receptor complex isgreater. This sharing of receptor subunits is similar to the shared useof the common gamma chain by cytokines such as IL2, IL4, IL7, IL9, andIL15. Some cell lines that do not respond to IL10 respond to IL-TIF byactivation of STAT-1, STAT-3, and STAT-5.

[0228] A soluble secreted receptor (231 amino acids), designated IL22BP[IL22 binding protein] has been described (Kotenko et al). The proteindemonstrates 34 percent amino acid identity with the extracellulardomain of the IL22R1 chain and is known also as CRF2-10. The gene mapsto human chromosome 6q24, 35 kb from the IFN-gamma R1 gene. It isexpressed in various tissues with maximal expression in breast, lungs,and colon. The protein binds IL-TIF and inhibits its activity, blockingits interaction with the cell surface IL22 receptor complex and thusacting as a natural cytokine antagonist. IL22BP also blocks induction ofthe suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL22in HepG2 cells.

[0229] References: Dumoutier L et al Cloning and characterization ofIL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokinestructurally related to IL-10 and inducible by IL-9. Journal ofImmunology 164(4): 1814-1819 (2000); Dumoutier L et al Humaninterleukin-10-related T cell-derived inducible factor: molecularcloning and functional characterization as an hepatocyte-stimulatingfactor. Proceedings of the National Academy of Science (USA) 97(18):10144-9 (2000); Dumoutier L et al IL-TIF/IL-22: genomic organization andmapping of the human and mouse genes. Genes Immun 1(8): 488-494 (2000);Dumoutier L et al Cloning and characterization of il-22 binding protein,a natural antagonist of il-10-related t cell-derived induciblefactor/il-22. Journal of Immunology 166(12): 7090-5 (2001); Kotenko S Vet al Identification, cloning, and characterization of a novel solublereceptor that binds IL-22 and neutralizes its activity. Journal ofImmunology 166(12): 7096-7103 (2001); Kotenko S V et al Identificationof the functional interleukin-22 (IL-22) receptor complex: the IL-10R2chain (IL-10 Rbeta) is a common chain of both the IL-10 and IL-22(IL-10-related T cell-derived inducible factor, IL-TIF) receptorcomplexes. Journal of Biological Chemistry 276(4): 2725-32 (2001); Xie MH et al Interleukin (IL)-22, a novel human cytokine that signals throughthe interferon receptor-related proteins CRF2-4 and IL-22R. Journal ofBiological Chemistry 275(40): 31335-9 (2000).

[0230] IL-23 is the name given to a factor that is composed of the p40subunit of IL12 (IL12B) and another protein of 19 kDa, designated p19.p19 is structurally related to IL6, G-CSF, and the p35 subunit of IL12.In databanks the p19 subunit is found also under the acronym SGRF (IL6G-CSF related factor).

[0231] 19 by itself is biologically inactive while the complex of p19with p40 is active. The active complex is secreted by dendritic cellsafter cell activation.

[0232] Mouse memory T-cells (CD4 (+) CD45 Rb(low)) proliferate inresponse to IL23 but not in response to IL12. Human IL23 has been shownto stimulate the production of IFN-gamma by PHA blast T-cells and memoryT-cells. It also induces proliferation of both cell types.

[0233] IL23 binds to the beta-i subunit but not to the beta-2 subunit ofthe IL12 receptor, activating one of the STAT proteins, STAT4, in PHAblast T-cells.

[0234] Expression of p19 in transgenic mice leads to runting, systemicinflammation, infertility, and death before 3 months of age. The animalsshow high serum concentrations of the pro-inflammatory cytokinesTNF-alpha and IL1. The number of circulating neutrophils is increased.Acute phase proteins are expressed constitutively. Animals expressingp19 specifically in the liver do not show these abnormalities.Expression of p19 is most likely due to hematopoietic cells as bonemarrow transplantation of cells expressing p19 causes the same phenotypeas that observed in the transgenic animals.

[0235] References: Oppmann B et al Novel p19 protein engages IL-12p40 toform a cytokine, IL-23, with biological activities similar as well asdistinct from IL-12. Immunity 13(5): 715-25 (2000); Wiekowski M T et alUbiquitous Transgenic Expression of the IL-23 Subunit p19 InducesMultiorgan Inflammation, Runting, Infertility, and Premature Death.Journal of Immunology 166(12): 7563-70 (2001).

[0236] IL24 is a name given to a protein that is known also as ST16[suppression of tumorigenicity-16] and MDA-7 [melanomadifferentiation-associated gene 7]. The rat counterpart of IL24 has beenidentified as mob-5 or C49a. The murine counterpart is FISP.

[0237] MDA-7 protein (206 amino acids) was identified initially as amelanoma differentiation-associated cDNA in a study using cultured humanmelanoma cells that lose proliferative capacity and terminallydifferentiate in response to human IFN-beta and mezerein. The expressionof MDA-7 is upregulated as a consequence of terminal differentiation.H0-1 and C8161 human melanoma cells engineered to express MDA-7 showreduces growth and do not form colonies in a colony formation assay.MDA-7 selectively suppresses the growth of human breast cancer cells bypromoting cell death by apoptosis. Ectopic expression of MDA-7 by meansof a replication defective adenovirus results in growth suppression andinduction of apoptosis in a broad spectrum of additional cancers,including melanoma, glioblastoma multiforme, osteosarcoma and carcinomasof the breast, cervix, colon, lung, nasopharynx and prostate. Noapparent harmful effects are observed after expression of MDA-7 innormal epithelial or fibroblast cells.

[0238] In human hematopoietic cells MDA-7 expression is induced duringmegakaryocyte differentiation in response to treatment with TPA(12-O-tetradecanoyl-phorbol-13-acetate).

[0239] The human MDA-7 gene maps to chromosome 1q32 and is tightlylinked (within a region of 195 kb) to the genes encoding IL10, IL19, andIL20.

[0240] The receptor for IL24 has been identified as the IL20 receptorcomplex. This receptor also binds to IL19.

[0241] References: Blumberg H et al Interleukin 20: discovery, receptoridentification, and role in epidermal function. Cell 104(1): 9-19(2001); Dumoutier L et al Cutting edge: STAT activation by IL-19, IL-20and mda-7 through IL-20 receptor complexes of two types. Journal ofImmunology 167(7): 3545-9 (2001); Huang EY et al Genomic structure,chromosomal localization and expression profile of a novel melanomadifferentiation associated (mda-7) gene with cancer specific growthsuppressing and apoptosis inducing properties. Oncogene 20(48): 7051-63(2001); Jiang H et al Subtraction hybridization identifies a novelmelanoma differentiation associated gene, mda-7, modulated during humanmelanoma differentiation, growth and progression. Oncogene 11: 2477-2486(1995); Jiang H et al The melanoma differentiation associated gene mda-7suppresses cancer cell growth. Proceedings of the National Academy ofScience (USA) 93: 9160-9165 (1996); Su Z et al The cancer growthsuppressor gene mda-7 selectively induces apoptosis in human breastcancer cells and inhibits tumor growth in nude mice. Proceedings of theNational Academy of Science (USA) 95: 14400-14405 (1998).

[0242] IL25 (also known as SF20) has been identified in a search forfactors that stimulate cell proliferation. The factor is secreted bybone marrow stromal cells.

[0243] The IL25 receptor has been identified as mouse thymic sharedantigen-1 (TSA-1). Enforced expression of the receptor in one of thefactor-dependent cell lines , BaF3, which does not express the receptor,causes cell proliferation. FDCP2 cells, which express the receptor, alsoproliferate in response to SF20/IL25. In both cases proliferation isabolished by specific blocking antibodies directed against the receptor.

[0244] SF20/IL-25 has no detectable myelopoietic activity but supportsproliferation of cells in the lymphoid lineage.

[0245] References: Tulin E E et al SF20/IL-25, a Novel Bone MarrowStroma-Derived Growth Factor That Binds to Mouse Thymic Shared Antigen-1and Supports Lymphoid Cell Proliferation. Journal of Immunology 167(11):6338-47 (2001)

[0246] The members of the TNF ligand superfamily (TNFalpha, TNF-beta, LTbeta, CD27 ligand, CD30 ligand, CD40 ligand, CD95 ligand, 4 1BB, OX40ligand, TRAIL) share common biological activities, but some propertiesare shared by only some ligands, while others are unique. HumanTNF-alpha is a non-glycosylated protein of 17 kDa and a length of 157amino acids. Murine TNF-alpha is N-glycosylated. Homology with TNF-betais approximately 30%. TNF-alpha forms dimers and trimers. The 17 kDaform of the factor is produced by processing of a precursor protein of233 amino acids. A TNF-alpha converting enzyme has been shown to mediatethis conversion. A transmembrane form of 26 kDa has been described also.

[0247] TNF-alpha contains a single disulfide bond that can be destroyedwithout altering the biological activity of the factor. Mutations Ala84to Val and Val91 to Ala reduce the cytotoxic activity of the factoralmost completely. These sites are involved in receptor binding. Thedeletion of 7 N-terminal amino acids and the replacement ofPro8Ser9Asp10 by ArgLysArg yields a mutated factor with an approximately10-fold enhanced antitumor activity and increased receptor binding, asdemonstrated by the L-M cell assay, while at the same time reducing thetoxicity.

[0248] The gene has a length of approximately 3.6 kb and contains fourexons. The primary transcript has a length of 2762 nucleotides andencodes a precursor protein of 233 amino acids. The aminoterminal 78amino acids function as a presequence. The human gene maps to chromosome6p23-6q12. It is located between class I HLA region for HLA-B and thegene encoding complement factor C. The gene encoding TNF-beta isapproximately 1.2 kb downstream of the TNF-alpha gene. However, bothgenes are regulated independently. The two genes also lie close to eachother on murine chromosome 17.

[0249] Approximately 500-10000 high-affinity receptors (Ka=2.5×10⁻⁹ M)for TNF-alpha are expressed on all somatic cell types with the exceptionof erythrocytes. Two receptors of 55 kDa (TNF-R1; new designation:CD120a) (e.g. polypeptides encoded by Genbank Accession No. X55313) and75 kDa (TNF-R2; new designation: CD120b) (e.g. as described in Goodwin RG et al (1991) Molecular Cellular Biology 11: 3020-6) have beendescribed. One receptor is a glycosylated protein of 455 amino acidsthat contains an extracellular domain of 171 and a cytoplasmic domain of221 amino acids. Sequence homologies in the cysteine-rich domains of theextracellular portion reveal that the receptor is related to thelow-affinity receptor of NGF and to human cell surface antigen CD40.

[0250] Deletion analysis in the C-terminal intracellular region of the55 kDa receptor, TNF-R1 has revealed the existence of a so-called deathdomain, which is involved in signaling processes leading to programmedcell death. The death domain of TNF-R1 interacts with a variety of othersignaling adaptor molecules, including TRADD, and RIP.

[0251] The two known receptors bind both TNF-alpha and TNF-beta. p55 isexpressed particularly on cells susceptible to the cytotoxic action ofTNF. p75 is also present on many cell types, especially those of myeloidorigin (a virus-encoded homologue of the receptor subunit is EBV-inducedgene-6). It is strongly expressed on stimulated T-cells andB-lymphocytes. The differential activities of TNF on various cell types,i. e. growth-promoting and growth-inhibiting activities, are probablymediated by the differential expression and/or regulation of multiplereceptors in combination with other distinct receptor-associatedproteins. p55 appears to play a critical role in host defenses againstmicroorganisms and their pathogenic factors.

[0252] A third receptor subtype is expressed in normal human liver. Itbinds TNF-alpha but not TNF-beta. Some viruses contain genes encodingsecreted proteins with TNF binding properties that are closelyhomologous to the p55 and p75 TNF receptors. Differential effects of thetwo receptor subtypes have been found also in TNF-mediated adhesion ofleukocytes to the endothelium. It appears that engagement of the p55receptor specifically leads to the induction of the cellular adhesionmolecules ICAM-1, E-selectin, V-CAM-1, and CD44, while engagement ofboth the p55 and the p75 receptor induces expression of alpha-2integrin.

[0253] Truncated soluble forms of the receptor have been found also. Thesoluble forms, in particular the soluble extracellular domain of the p60receptor, block the antiproliferative effects of TNF and, therefore, maymodulate the harmful effects of TNF.

[0254] Receptor densities are reduced by IL1 and tumor promoters such asphorbol esters. The expression of TNF-alpha receptor density is inducedby IFN-alpha, IFN-beta, and IFN-gamma.

[0255] Signal transducers that associate with the cytoplasmic domains ofmembers of the TNF receptor superfamily comprise TRAF (Tumor necrosisfactor receptor-associated factors).

[0256] Human TNF-alpha is active on murine cells with a slightly reducedspecific activity. In general, TNF-alpha and TNF-beta display similarspectra of biological activities in in-vitro systems, although TNF-betais often less potent or displays apparent partial agonist activity.

[0257] TNF-alpha shows a wide spectrum of biological activities. Itcauses cytolysis and cytostasis of many tumor cell lines in vitro.Sensitive cells die within hours after exposure to picomolarconcentrations of the factor and this involves, at least in part,mitochondria-derived second messenger molecules serving as commonmediators of TNF cytotoxic and gene-regulatory signaling pathways. Thefactor induces hemorrhagic necrosis of transplanted tumors. Within hoursafter injection TNF-alpha leads to the destruction of small bloodvessels within malignant tumors. The factor also enhances phagocytosisand cytotoxicity in neutrophilic granulocytes and also modulates theexpression of many other proteins, including fos, myc, IL1 and IL6.

[0258] The 26 kDa form of TNF is found predominantly on activatedmonocytes and T-cells. It is also biologically active and mediates celldestruction by direct cell-to-cell contacts.

[0259] The chemotactic properties of fMLP (Formyl-Met—Leu—Phe) forneutrophils are enhanced by TNF-alpha. TNF-alpha induces the synthesisof a number of chemoattractant cytokines, including IP-10, JE, KC, in acell-type and tissue-specific manner.

[0260] TNF-alpha is a growth factor for normal human diploidfibroblasts. It promotes the synthesis of collagenase and prostaglandinE2 in fibroblasts. It may also function as an autocrine growth modulatorfor human chronic lymphocytic leukemia cells in vivo and has beendescribed to be an autocrine growth modulator for neuroblastoma cells.The autocrine growth-promoting activity is inhibited by IL4.

[0261] In resting macrophages TNF induces the synthesis of IL1 andprostaglandin E2. It also stimulates phagocytosis and the synthesis ofsuperoxide dismutase in macrophages. TNF activates osteoclasts and thusinduces bone resorption.

[0262] In leukocyte and lymphocyte progenitors TNF stimulates theexpression of class I and II HLA and differentiation antigens, and theproduction of IL1, colony stimulating factors, IFN-gamma, andarachidonic acid metabolism. It also stimulates the biosynthesis ofcollagenases in endothelial cells and synovial cells.

[0263] IL6 suppresses the synthesis of IL1 induced by bacterialendotoxins and TNF, and the synthesis of TNF induced by endotoxins.

[0264] The neurotransmitter SP (substance P) induces the synthesis ofTNF and IL1 in macrophages. IL1, like IL6, stimulates the synthesis ofACTH (corticotropin) in the pituitary. Glucocorticoids synthesized inresponse to ACTH in turn inhibit the synthesis of IL6, IL1 and TNF invivo, thus establishing a negative feedback loop between the immunesystem and neuroendocrine functions.

[0265] TNF-alpha enhances the proliferation of T-cells induced byvarious stimuli in the absence of IL2. Some subpopulations of T-cellsonly respond to IL2 in the presence of TNF-alpha. In The presence of IL2TNF-alpha promotes the proliferation and differentiation of B-cells.

[0266] The functional capacities of skin Langerhans cells are alsoinfluenced by TNF-alpha. These cells are not capable of initiatingprimary immune responses such as contact sensibilisation. They areconverted into immunostimulatory dendritic cells by GM-CSF and also IL1.These cells therefore are a reservoir for immunologically immaturelymphoid dendritic cells. The enhanced ability of maturated Langerhanscells to process antigens is significantly reduced by TNF-alpha.

[0267] Although TNF-alpha is also required for normal immune responsesthe overexpression has severe pathological consequences. TNF-alpha isthe major mediator of cachexia observed in tumor patients (hence itsname, cachectin). TNF is also responsible for some of the severe effectsduring Gram-negative sepsis.

[0268] TNF-alpha can be detected in bioassays involving cell lines thatrespond to it (e.g., BT-20, CT6, EL4; PK15; L929; L-M ; MO7E; T1165;WEHI-3B). TNF-alpha can be detected also by a sensitive sandwich enzymeimmunoassay, ELISA, an immunoradiometric assay (IRMA), and by an assaydesignated RELAY (receptor-mediated label-transfer assay). Intracellularfactor is detected by two color immunofluorescence flow cytometry.Higuchi et al have described an assay based on the release of tritiatedthymidine from cells undergoing apoptosis after treatment with eitherTNF-alpha or TNF-beta. IFN-alpha, IFN-beta, IFN-gamma, TGF-beta, IL4,LIF and GM-CSF have been shown not to interfere with this assay.

[0269] In contrast to chemotherapeutic drugs TNF specifically attacksmalignant cells. Extensive preclinical studies have documented a directcytostatic and cytotoxic effect of TNF-alpha against subcutaneous humanxenografts and lymph node metastases in nude mice, as well as a varietyof immunomodulatory effects on various immune effector cells, includingneutrophils, macrophages, and T-cells. Single- and multiple-dose phase Istudies have confirmed that TNF can be administered safely to patientswith advanced malignancies in a dose range associated with anticancereffect without concomitant serious toxicities such as shock andcachexia. However, clinical trials on the whole have unfortunately sofar failed to demonstrate significant improvements in cancer treatment,with TNF-induced systemic toxicity being a major limitation for the useof TNF as an antineoplastic agent in most cases. The combined use of TNFand cytotoxic or immune modulatory agents, particularly IFN-gamma andpossibly IL2, may be of advantage in the treatment of some tumors. Insome cases intratumoral application of TNF has been found to be ofadvantage in tumor control.

[0270] Some mutant forms of TNF-beta with selective activity on the p55receptor have been described recently. It has been shown that activationof the p55 receptor is sufficient to trigger cytotoxic activity towardstransformed cells. Some of these mutants have been described to retaintheir antitumor activity in nude mice carrying transplanted humantumors.

[0271] TNF can also be used to increase the aggressiveness oflymphokine-activated killer cells. Studies with an experimentalfibrosarcoma metastasis model have shown that TNF induces significantenhancement of the number of metastases in the lung. It has beensuggested that low doses of endogenous TNF or administration of TNFduring cytokine therapy may enhance the metastatic potential ofcirculating tumor cells. The transduction of murine tumor cells with afunctional TNF-alpha gene has been shown to lead to the rejection of thegenetically modified cells by syngeneic hosts.

[0272] The interferons are a family of cytokines that induce avirus-nonspecific antiviral state in target cells. Binding of aninterferon to its receptor induces new protein synthesis which, in turn,results in the inactivation of initiation factor eIF-2. The inactivationis thought to contribute to the antiviral state induced by theinterferons. Interferons also induce pathways that activateintracellular endonucleases which degrade viral mRNA. Many interferonsalso possess immunomodulatory activities, such as activation ofmacrophages and lymphocytes. Examples of interferons include IFN-gamma(e.g. polypeptides encoded by Genbank Accession No. K01900, J00209,M12350, J00213, J00216, J00214, M11003, M11026, M34913, M54886, X01974,L38698, M13710, K01238, M13660, M68944, X01972, X01971, X01973, X01969),IFN-gamma (e.g. polypeptides encoded by Genbank Accession No.M28622,X14029, X14455, K00020, J00218, E00171, X04430, A09363, M27327, M16656,M25460, K03196), IFN-gamma e.g. polypeptides encoded by GenbankAccession No. A34532, X87308, E00756, K00083), IFN-gamma e.g.polypeptides encoded by Genbank Accession No. X58822, A12140), bovinetrophoblast protein-1 (IFN-gamma) e.g. polypeptides encoded by GenbankAccession No. M31556, M31557, M31558), and their homologues amongspecies. Human IFN-gamma and IFN-gamma are thought to bind to a commonreceptor (e.g. polypeptides encoded by Genbank Accession No. X60459,M89641) which is distinct from the receptor for IFN-gamma (e.g.polypeptides encoded by Genbank Accession No. J03143, M28233).

[0273] At least 23 different variants of IFN-alpha are known. Theindividual proteins have molecular masses between 19-26 kDa and consistof proteins with lengths of 156-166 and 172 amino acids. All IFN-alphasubtypes possess a common conserved sequence region between amino acidpositions 115-151 while the amino-terminal ends are variable. ManyIFN-alpha subtypes differ in their sequences at only one or twopositions. Naturally occurring variants also include proteins truncatedby 10 amino acids at the carboxy-terminal end. Disulfide bonds areformed between cysteines at positions 1/98 and 29/138. The disulfidebond 29/138 is essential for biological activity while the 1/98 bond canbe reduces without affecting bioactivity.

[0274] Human IFN-beta is a glycoprotein (approximately 20% sugar moiety)of 20 kDa and has a length of 166 amino acids. Glycosylation is notrequired for biological activity in vitro. The protein contains adisulfide bond Cys31/141) required for biological activity. At the DNAlevel IFN-beta displays 34% sequence homology with IFN-beta-2 andapproximately 30% homology with other IFN-alpha subtypes. In contrast toIFN-gamma IFN-beta is stable at pH2.

[0275] Human IFN-gamma is a dimeric protein with subunits of 146 aminoacids. The protein is glycosylated at two sites. The pI is 8.3-8.5.IFN-gamma is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-gamma with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-gamma.

[0276] Members of the CSF family of cytokines allow the growth anddifferentiation of bone marrow cells immobilized on soft agar ormethylcellulose. While hematopoietic progenitor cells can be maintainedonly for short periods of time in the absence of such factors, theirpresence allows the development of colonies containing erythroid cells,neutrophils, eosinophils, macrophages, and/or megakaryocytes, dependingon the particular factor. The biochemical analysis of various activitiesstimulating colony formation supporting the growth and development ofthese cell types revealed that there existed many different and distinctfactors of this sort.

[0277] Many of these factors are either N- or O-glycosylated.Glycosylation has been shown to enhance the solubility, stability andresistance to proteolytic enzymes. It does not appear to be required forthe full spectrum of biological activities of these factors. The genesencoding many of the human colony stimulating factors have been clonedand mapped. Some of the genes are in close vicinity but they do not showgreat homology among each other with the exception of some conservedregions.

[0278] Colony stimulating factors are produced by many different celltypes, including, for example, B-lymphocytes, epithelial cells,fibroblasts, endothelial cells, macrophages, Stromal cell line,T-lymphocytes. They are synthesized as precursor molecules containing aclassical hydrophobic secretory signal sequence of approximately 25-32amino acids. The secreted factors have an extremely high specificbiological activity are active at very low concentrations (1-100 pM).These factors are absolutely required for the proliferation ofhematopoietic progenitor cells. The concentrations required for meremaintenance of viability are usually orders of magnitude lower thanthose required to induce cell proliferation or to elicit specificfunctional activities of the cells.

[0279] The names of the individual factors usually indicate the celltypes that respond to these factors. The classical colony stimulatingfactors include M-CSF (e.g. polypeptides encoded by Genbank AccessionNo. E03235, M64592, U22386, X05010) (macrophage-specific), G-CSF(granulocyte-specific), GM-CSF (macrophage/granulocyte-specific), IL3(multifunctional) and MEG-CSF (e.g. polypeptides encoded by GenbankAccession No.D86370, U70136) (megakaryocyte-specific). G-CSF and M-CSFare lineage-specific while GM-CSF and IL3 are multifunctionalhematopoietic growth factors acting on earlier stages of differentiationof hematopoietic progenitor cells.

[0280] Human GM-CSF is a monomeric protein of 127 amino acids with twoglycosylation sites. The protein is synthesized as a precursor of 144amino acids, which included a hydrophobic secretory signal sequence atthe aminoterminal end. The sugar moiety is not required for the fullspectrum of biological activities. Non-glycosylated and glycosylatedGM-CSF show the same activities in vitro. Fully glycosylated GM-CSF isbiologically more active in vivo than the nonglycosylated protein. Thedifferent molecular weight forms of GM-CSF (14 kDa, 35 kDa) described inthe literature are the result of varying degrees of glycosylation.GM-CSF contains four cysteine residues (positions 54/96 and 88/121).

[0281] A comparison of the protein sequence of GM-CSF with those of theother colony stimulating factors reveals that they are not stronglyhomologous to each other. Human and murine GM-CSF display 60% homologyat the protein level and 70% at the nucleotide level. The two factors donot, however, cross-react immunologically. GM-CSF can be associated withthe extracellular matrix of cells as a complex with heparan sulfateproteoglycans. This allows storage of the factor in a biologicallyinactive form. The exact mechanism by which the factor is eventuallyreleased from these depots is not known.

[0282] The human gene has a length of approximately 2.5 kb and containsfour exons. The distance between the GM-CSF gene and the IL3 gene isapproximately 9 kb. The human GM-CSF gene maps to chromosome 5q22-31 inthe vicinity of other genes encoding hematopoietic growth factors(M-CSF, IL3, IL4, IL5) and the gene encoding the M-CSF receptor. The 5′region of the GM-CSF gene contains several sequence elements known asCLE (conserved lymphokine element). They function as binding sites fortranscription factors, modulating the expression of the GM-CSF gene.

[0283] GM-CSF receptors are expressed at densities of several 100 toseveral 1000 copies/cell on the cell surface of myeloid cells. Thereceptor is expressed also on non-hematopoietic cells such asendothelial cells and small cell lung carcinoma cells. Inreceptor-positive cell lineages the receptor density decreases withincreasing degrees of maturation.

[0284] The receptor shows significant homologies with other receptorsfor hematopoietic growth factors, including IL2-beta, IL3, IL6, IL7, Epoand the prolactin receptors. One cloned subunit of the GM-CSF receptor(GM-R alpha, 45 kDa) binds GM-CSF with low affinity (e.g. polypeptidesencoded by Genbank Accession No. SEG_HUMGRAS). The second subunit (GM-Rbeta, 120 kDa) does not bind GM-CSF. GM-R alpha is a protein of 400amino acids that contains only a short cytoplasmic domain of 54 aminoacids. The high affinity GM-CSF receptor is formed by the aggregation ofthe two receptor subunits. The GM-R beta subunit of the receptor (e.g.polypeptides encoded by Genbank Accession No. SEG_MUSAIC2B, M59941) isalso a constituent of other cytokine receptor systems. It is a componentof the high affinity receptors for IL3 and IL5, both of which alsocontain a cytokine-specific subunit (AIC2A).

[0285] Human GM-CSF is not active on murine cells and vice versa. GM-CSFwas isolated initially as a factor stimulating the growth ofmacrophage/granulocyte-containing colonies in soft agar cultures (colonyformation assay). GM-CSF is indispensable for the growth and developmentof granulocyte and macrophage progenitor cells. It stimulatesmyeloblasts and monoblasts and triggers irreversible differentiation ofthese cells. GM-CSF synergises with Epo in the proliferation oferythroid and megakaryocytic progenitor cells. In combination withanother colony stimulating factor, M-CSF, one observes the phenomenon ofsynergistic suppression, i.e., the combination of these two factorsleads to a partial suppression of the generation ofmacrophage-containing cell colonies.

[0286] For some types of blast cells from patients with acute myeloidleukemia GM-CSF acts as an autocrine mediator of growth. GM-CSF is astrong chemoattractant for neutrophils. It enhances microbicidalactivity, oxidative metabolism, and phagocytotic activity of neutrophilsand macrophages. It also improves the cytotoxicity of these cells.GM-CSF displays a less pronounced specificity than, for example, G-CSF.It stimulates the proliferation and differentiation of neutrophilic,eosinophilic, and monocytic lineages. It also functionally activates thecorresponding mature forms, enhancing, for example, the expression ofcertain cell surface adhesion proteins (CD-11A, CD-11C). Theoverexpression of these proteins could be one explanation for theobserved local accumulation of granulocytes at sites of inflammation. Inaddition, GM-CSF also enhances expression of receptors for fMLP(Formyl-Met—Leu—Phe) which is a stimulator of neutrophil activity.

[0287] At pico to nanomolar concentrations GM-CSF is chemotactic foreosinophils and also influences the chemotactic behavior of these cellsin response to other chemotactic factors.

[0288] In granulocytes GM-CSF stimulates the release of arachidonic acidmetabolites and the increased generation of reactive oxygen species. Theactivation of the Na+/H+ antiport system leads to a rapid alkalizationof the cytosol. Phagocytotic activities of neutrophil granulocytes andthe cytotoxicity of eosinophils is also enhanced considerably by GM-CSF.Since GM-CSF is produced by cells (T-lymphocytes, tissue macrophages,endothelial cells, mast cells) present at sites of inflammatoryresponses it can be assumed that it is an important mediator forinflammatory reactions.

[0289] The functional state of Langerhans cells of the skin is alsoinfluenced by GM-CSF. These cells are not capable of initiating primaryimmune responses, for example, contact sensibilization. They areconverted to highly potent immunostimulatory dendritic cells by GM-CSF(and also IL1). Langerhans cells therefore form an in situ reservoir forimmunologically immature lymphoid dendritic cells. The maturation ofthese cells which is seen as an increased ability to process antigens,can be down-regulated by TNF-alpha.

[0290] At nanomolar concentrations GM-CSF induces the expression ofcomplement C3a receptors on basophils. Cells which normally do notrespond to C3a and which have been activated by GM-CSF degranulate inresponse to the C3a stimulus. This is accompanied by the release ofhistamine and leukotriene C4. This process may be of significance inhypersensitivity reactions associated with inflammatory responses(T-lymphocytes, tissue macrophages, endothelial cells, mast cells).GM-CSF has been shown also to be a potent inducer of trophoblastinterferon (TP-1).

[0291] GM-CSF synergises with some other cytokines, including IL1, IL3and G-CSF. GM-CSF and G-CSF must act in concert to allow the developmentof neutrophil-containing colonies in vitro.

[0292] IL3 by itself only negligibly expands the number of circulatingblood cells; a subsequent dose of GM-CSF, however, significantlyincreases cell numbers, probably because IL3 first leads to an expansionof those cells capable of responding to GM-CSF.

[0293] The observations that most IL3-dependent cell lines can also growin the presence of GM-CSF and IL4 and that several synergistic effectsare observed between GM-CSF and IL4 suggest that these three factorsperform similar functions in controlling the growth of cells. There aresome indications that the mechanism of signal transduction contains atleast some common factors.

[0294] Experiments with tyrosine-specific protein kinases encoded by anoncogene have shown that the expression of this kinase activity infactor-dependent cells abolishes their dependence on GM-CSF, IL3 andIL4. The exact mechanism by which these factors regulate theproliferation and differentiation of cells is still unknown.

[0295] The consequences of a deregulated expression of GM-CSF have beenstudied in transgenic mice harboring a constitutively expressed GM-CSFgene. The overexpression of the transgene encoding GM-CSF leads topathological alterations in the retina and causes blindness and alsocauses muscle deterioration. These mice are characterized by a verypronounced increase in activated macrophages. In addition, theoverexpression of GM-CSF leads to the activation of mature macrophagessecreting large amounts of IL1 and TNF, suggesting that these cytokinesmay be responsible for some aspects of the transgenic mouse disease.

[0296] Histopathological examination demonstrates a pronounced increasein the progenitor cell population of the monocytic lineage.GM-CSF-transgenic animals usually die within months from the massivetissue damage resulting from the overexpression of these factors.Similar results have been obtained with mice possessing a bone marrowmanipulated to overexpress GM-CSF by transformation with suitableretrovirus vectors. These findings do not seem to be of clinicalsignificance, though. The long-term treatment of primates and mice withGM-CSF has shown that life-threatening complications do not occur.

[0297] The biological consequences of GM-CSF gene disruption have beenstudied in mice generated from ES cells carrying a targeted deletion ofthe gene. Mice homozygous for a targeted disruption of the GM-CSF geneare characterized by an unimpaired steady-state hematopoiesis,demonstrating that GM-CSF is not essential for maintaining normal levelsof the major types of mature hematopoietic cells and their precursors inblood, marrow, and spleen.

[0298] Most GM-CSF-deficient mice are superficially healthy and fertilebut develop abnormal lungs. GM-CSF-deficient mice develop a progressiveaccumulation of surfactant lipids and proteins in the alveolar space,the defining characteristics of the idiopathic human disorder pulmonaryalveolar proteinosis. Extensive lymphoid hyperplasia associated withlung airways and blood vessels is found also. These results demonstratean unexpected, critical role for GM-CSF in pulmonary homeostasis.

[0299] Transgenic mice homozygous for null mutations of the geneencoding the common beta subunit (beta C) of the GM-CSF, IL3, and IL5receptor complexes exhibit normal development and survive to young adultlife. They develop pulmonary peribronchovascular lymphoid infiltratesand areas resembling alveolar proteinosis. Eosinophil numbers inperipheral blood and bone marrow of homozygous deletion mutants arereduced, while other hematological parameters are normal. Bone marrowcells from homozygous deletion mutants do not show high-affinity bindingof GM-CSF, while cells from heterozygous animals show an intermediatenumber of high-affinity receptors. In clonal cultures of bone marrowcells derived from homozygous deletion mutants, even high concentrationsof GM-CSF and IL5 do not stimulate colony formation in the colonyformation assay. Differences in the systemic clearance and distributionof GM-CSF between mutant and wild-type littermates are not observed.

[0300] Nishinakamura et al have crossed beta-c mutant mice with micedeficient for IL3. The double-mutant mice lacking all IL3, GM-CSF, andIL5 functions are apparently normally fertile. The animals show the samereduced numbers of eosinophils and a lack of eosinophilic response toparasites as beta-c mutant mice. The immune response of the doublemutant mice to Listeria monocytogenes is normal. Hematopoietic recoveryafter treatment with fluorouracil is also normal. These findings suggestthe existence of alternative mechanism to produce blood cells that donot depend on the presence of IL3, GM-CSF, and IL5.

[0301] GM-CSF can be assayed in a colony formation assay by thedevelopment of colonies containing macrophages, neutrophils,eosinophils, and megakaryocytes. GM-CSF is also detected in specificbioassays with cells lines that depend in their growth on the presenceof GM-CSF or that respond to this factor (e.g., AML-193; B6SUt-A;BAC1.2F5; BCL1; Da; FDCP1; GF-D8; GM/SO; IC-2; MO7E; NFS-60; PT-18;TALL-103; TF-1; UT-7).

[0302] GM-CSF can be employed for the physiological reconstitution ofhematopoiesis in all diseases characterized either by an aberrantmaturation of blood cells or by a reduced production of leukocytes. Themain and most important clinical application of GM-CSF is probably thetreatment of life-threatening neutropenia following chemo and/orradiotherapy, which is markedly reduced under GM-CSF treatment. GM-CSFcan be used also to correct chemotherapy-induced cytopenias and tocounteract cytopenia-related predisposition to infections andhemorrhages.

[0303] In order to avoid potential complications following theadministration of GM-CSF careful clinical monitoring is required incertain patient groups, for example those with myelodysplastic syndrome,acute myeloid leukemia, inflammatory disease, autoimmunethrombocytopenia or malfunctional immunological responsiveness.

[0304] Several studies have demonstrated that the use of GM-CSF enhancestolerance to cytotoxic drug treatment and can be used to prevent dosereductions necessitated by the side effects of cytotoxic drug treatment.GM-CSF treatment frequently permits to increase the doses of cytotoxicdrugs per course. These studies have also revealed a significantlyreduced morbidity under GM-CSF treatment.

[0305] The transduction of murine tumor cells with a functional GM-CSFgene has been shown to lead to the rejection of the genetically modifiedcells by syngeneic hosts. Moreover, vaccination with GM-CSF transducedtumor cells prevents growth of a subsequent inoculum of wild-typesyngeneic tumor cells.

[0306] The chemokine family of cytokines consists of relatively small,structurally similar polypeptides that induce chemotaxis in leukocytes.Chemokines have molecular masses of 8-10 kDa and show approximately20-50% sequence homology among each other at the protein level. Theproteins also share common gene structures and tertiary structures. Allchemokines possess a number of conserved cysteine residues involved inintramolecular disulfide bond formation.

[0307] According to the chromosomal locations of individual genes twodifferent subfamilies of chemokines are distinguished. Members of thealpha-chemokines are referred to also as the 4q chemokine family becausethe genes encoding members of this family map to human chromosome 4q12-21. The first two cysteine residues of members of this family areseparated by a single amino acids and these proteins, therefore, arecalled also C-X-C chemokines. This subfamily includes 9E3 (e.g. Genbankprotein P08317), AMCF (e.g. polypeptides encoded by Genbank AccessionNo. M99367, M99368), beta-thromboglobulin (e.g. as disclosed in Begg GSet al (1978), Biochemistry 17: 1739-44), CINC family members (e.g.polypeptides encoded by Genbank Accession No. D21095), ENA-78 (e.g.polypeptides encoded by Genbank Accession No. X78686), eotaxin (e.g.polypeptides encoded by Genbank Accession No. U46572, U40672), GCP-2(e.g. polypeptides encoded by Genbank Accession No. Y08770, U83303),IL8, IP-10 (e.g. polypeptides encoded by Genbank Accession No. L07417,X02530), KC (e.g. polypeptides encoded by Genbank Accession No. J04596),LIX (e.g. polypeptides encoded by Genbank Accession No. U27267), mig(e.g. polypeptides encoded by Genbank Accession No. M34815, Z24725),MGSA (e.g. polypeptides encoded by Genbank Accession No. X12510), mob-1(e.g. polypeptides encoded by Genbank Accession No. U17035), NAP-2 (asdescribed in Clark-Lewis I et al (1991) Biochemistry 30: 3128-35, CohenA B et al (1992) American Journal of Physiology 263: L249-56), NAP-3 (asdescribed in: Schroder J M et al (1991) Journal of Experimental Medicine171: 1091-100), NAP-4 (as described in Schroder J M et al (1990)Biochemical and Biophysical Research Communications 172: 898-904), PBSF(SDF) (e.g. polypeptides encoded by Genbank Accession No. D21072,U16752, D50645), and PF4 (e.g. polypeptides encoded by Genbank AccessionNo. M25897).

[0308] IL8, MGSA, mouse KC, MIP-2 (e.g. polypeptides encoded by GenbankAccession No. X65647 and as described in Blum S et al Three humanhomologues of a murine gene encoding an inhibitor of stem cellproliferation. DNA Cell Biol. 9: 589-602 (1990); Clements J M et alBiological and structural properties of MIP-1 alpha expressed in yeast.Cytokine 4: 76-82 (1992); Devatelis G et al Cloning and characterizationof a cDNA for murine macrophage inflammatory protein (MIP), a novelmonokine with inflammatory and chemokinetic properties. Journal ofExperimental Medicine 167: 1939-44 (1988) (erratum in JEM 170: 2189(1989)); Farber J M A macrophage mRNA selectively induced bygamma-interferon encodes a member of the platelet factor 4 family ofcytokines. Proceedings of the National Academy of Science (USA) 87:5238-42 (1990); Haskill S et al Identification of three related humanGRO genes encoding cytokine functions. Proceedings of the NationalAcademy of Science (USA) 87: 7732-6 (1990); Poltorak AN et al (1995)Journal of Inflammation 45(3): 207-19; Rossi D L et al (1997) Journal ofImmunology 158(3): 1033-1036; Sherry B et al (1988) Journal ofExperimental Medicine 168: 2251-9; Tekamp-Olson P et al (1990) Journalof Experimental Medicine 172: 911-9; Wolpe S D et al (1989) Proceedingsof the National Academy of Science (USA) 86: 612-16; Wolpe S D et al(1989) FASEB Journal 3: 2565-73), NAP-2, ENA-78, and GCP-2 comprise asubgroup of the human C-X-C-chemokines defined by the conserved ELRsequence motif (glutamic-acidleucine-arginine) immediately preceding thefirst cysteine residue near the amino-terminal end. Chemokines with anELR sequence motif have been found to chemoattract and activateprimarily neutrophils. Chemokines without the ELR sequence motif appearto chemoattract and activate monocytes, dendritic cells, T-cells,NK-cells, B-lymphocytes, basophils, and eosinophils.

[0309] Members of the beta-chemokines or 17q chemokine family map tohuman chromosome 17q11-32 (murine chromosomel 1). The first two cysteineresidues are adjacent and, therefore, these proteins are called also C-Cchemokines. This subfamily includes ACT-2 (e.g. polypeptides encoded byGenbank Accession No. J04130), C10 (e.g. as described in Berger M S etal (1993) DNA Cell Biol. 12: 839-47; Berger M S et al (1996) 8:439-447), CCF18 (e.g. as described in Hara T et al (1995) Journal ofImmunology 155: 5352-8), DC-CK1 (e.g. as described in Adema G J et al(1997) Nature 387: 713-717), ELC (e.g. polypeptides encoded by GenbankAccession No. AB000887, AF059208), Eotaxin-2 (e.g. as described inForssmann U et al (1997) Journal of Experimental Medicine 185:2171-2176), Exodus (e.g. polypeptides encoded by Genbank Accession No.U64197, U88320, U88321, U88322), FIC (e.g. polypeptides encoded byGenbank Accession No. L04694), GDCF and GDCF-2 (e.g. as described inKuratsu J et al (1989) Journal of the National Cancer Institute 81:347-51; Yoshimura T et al (1989) Journal of Experimental Medicine 169:1449-59; Yoshimura T et al (1989) Journal of Immunology 142: 1956-62),HC-21 (e.g. as described in Chang H C & Reinherz E L (1989) EuropeanJournal of Immunology 19:1045-1051), HCC-1 (e.g. polypeptides encoded byGenbank Accession No. Z49270), I-309 (e.g. polypeptides encoded byGenbank Accession No. M57502), JE (e.g. polypeptides encoded by GenbankAccession No. AF058786, M28226), LAG-1 (lymphocyte activation gene-1)(e.g. polypeptides encoded by Genbank Accession No. X53683), LARCD86955), LD78 E03130, E03131, MARC (e.g. as described in Thirion S et al(1994) Biochemical and Biophysical Research Communications 201:493-499), MCAF M24545 and as described in Apella E et al (1990) Progressin Clinical and Biological Research 349: 405-17), MCP-1 (e.g.polypeptides encoded by Genbank Accession No. X14768), MCP-2 (e.g.polypeptides encoded by Genbank Accession No. Y16645), MCP-3 (e.g.polypeptides encoded by Genbank Accession No. X72308, S71251), MCP-4(e.g. polypeptides encoded by Genbank Accession No. X98306), MCP-5 (e.g.polypeptides encoded by Genbank Accession No. U50712), MIP (macrophageinflammatory protein) (e.g. polypeptides encoded by Genbank AccessionNo. U77180, U77035, U49513, M35590), MRP-2 (e.g. as described in Youn BS et al (1995) Journal of Immunology 155: 2661-7), RANTES SDF (e.g.polypeptides encoded by Genbank Accession No. M21121, M77747), TARC (e.gGenbank protein Accession No. Q92583).

[0310] In addition there are several other factors that are related tochemokines but that either have not been assigned yet to one of the twochemokine groups or that do not possess the classical features of eitherof the two chemokine groups (for example, ATAC (e.g. polypeptidesencoded by Genbank Accession No. X86474), Ltn (e.g. polypeptides encodedby Genbank Accession No. U15607, U23772), SCM-1 (e.g. polypeptidesencoded by Genbank Accession No. D63789, D63790, D43769). These havebeen referred to as C-type chemokines or gamma-chemokines.

[0311] Yet another group of chemokines has been identified thatcomprises neurotactin (e.g. polypeptides encoded by Genbank AccessionNo. AF010586, which is characterized by a CX(3)C cysteine signaturemotif The existence of clearly defined subgroups of chemokines on thebasis of structural and functional properties illustrates the importanceof chemoattractant diversity in the regulation of leukocyte movementthrough the body.

[0312] The biological activities of chemokines are mediated by specificreceptors and also by receptors with overlapping ligand specificitiesthat bind several of these proteins which always belong either to theC-C-chemokines or the group of C-X-C-chemokines. Chemokine receptorsbelong to the large group of G-protein-coupled seven transmembranedomain receptors which contain seven hydrophobic alpha-helical segmentsthat transverse the membrane. These receptors form a structurallyrelated group within the superfamily of G-protein-coupled receptorswhich mediate signalling via heterotrimeric G-proteins.

[0313] The receptors that bind C-X-C chemokines are designated CXCRfollowed by a number (e.g., CXCR-1 (e.g. polypeptides encoded by GenbankAccession No. L19591), CXCR-2 (e.g. polypeptides encoded by GenbankAccession No. M94582), CXCR-3 (e.g. polypeptides encoded by GenbankAccession No. X95876), CXCR-4 (e.g. polypeptides encoded by GenbankAccession No. D87747, AF025375) while those binding C-C chemokines aredesignated CCR followed by a number (e.g., CCR-1 (e.g. polypeptidesencoded by Genbank Accession No.L09230, U29678), CCR-2 (e.g.polypeptides encoded by Genbank Accession No. U29677, U95626), CCR-3(e.g. polypeptides encoded by Genbank Accession No. U51241), CCR-4 (e.g.polypeptides encoded by Genbank Accession No. X90862, X85740), CCR-5(e.g. polypeptides encoded by Genbank Accession No. U54994, U83327),CCR-6 (e.g. polypeptides encoded by Genbank Accession No. U95626), CCR-7(e.g. polypeptides encoded by Genbank Accession No. L31581), CCR-8 (e.g.polypeptides encoded by Genbank Accession No. Z98206, U45983). Viralchemokine receptor homologues include ECRF-3, EBI-1 (EBV-inducedgene-1), and US28.

[0314] It is now assumed that the combinatorial effects of multiplechemokines and other mediators are responsible for the cellularcomposition at inflammatory sites. In addition, many chemokines alsodirectly activate cells. Some of them activate granulocytes and/ormonocytes and cause respiratory bursts, degranulation, and the releaseof lysosomal enzymes. Others prime immune cells to respond tosub-optimal amounts of other inflammatory mediators. Yet others havebeen shown to be potent histamine releasing factors for basophils. Ithas been proposed that erythrocytes through their promiscuous chemokinereceptor play an important role in regulating the chemokine network.Chemokines bound to the erythrocyte receptor are known to beinaccessible to their normal target cells. This appears to provide asink for superfluous chemokines and may serve to limit the systemiceffects of these mediators without disrupting localized processes takingplace at the site of inflammation.

[0315] Certain C-C chemokines exhibit biological activities other thanmere chemotaxis. Some chemokines have been shown to be capable ofinducing the proliferation and activation of killer cells known as CHAK(C-C-chemokine-activated killer), which are similar to cells activatedby IL2.

[0316] Another particularly useful cytokine according to the inventionis flt-3 ligand (e.g.,polypeptides encoded by Genbank Accession Nos.U04806, U04807, U03858, L23636, U29874, U29875, U44024). This cytokinebinds to the flt-3 tyrosine kinase (e.g., polypeptides encoded byGenbank Accession Nos. Z26652, X59398). The human flt-3 ligand alsostimulates the proliferation of cells expressing murine flt-3 receptors.

[0317] The effects of flt-3 ligand are synergized by coexpression ofG-CSF, GM-CSF, M-CSF, IL3, PIXY-321, and SCF. In combination with SCFand IL3 flt-3 ligand can cause expansion of cells with the markerspectrum CD34 (+)CD38 (−). Alone flt-3 ligand supports the survival ofprecursor cell types in the lineage of blood-forming cells such asCFU-GM, CFU-GEMM, and the very primitive high proliferative potentialcolony-forming cells. flt-3 ligand only has marginal effects onerythroid and megakaryocyte progenitor cells.

[0318] In the mouse, flt-3 ligand potently enhances growth of varioustypes of progenitor/precursor cells in synergy with G-CSF, GM-CSF,M-CSF, IL3, IL6, IL7, IL11, IL12 and SCF. flt-3 ligand supports growthof LTC-IC (long-term culture-initiating cells). The ability of flt-3ligand to promote the survival of hematopoietic progenitor cells isabrogated by TGF-beta and counteracted by TNF-alpha.

[0319] A study of the expression of functional flt-3 receptor and theresponses to the ligand in AML (acute myeloid leukemia) and ALL (acutelymphoblastic leukemia) shows a considerable heterogeneity. BCP-ALL inparticular fails to proliferate in the presence of flt-3 ligand despitestrong expression of surface flt-3 receptor.

[0320] It has been shown that in patients with aplastic anemia and incancer patients with chemotherapy-induced transient suppression ofhematopoiesis, serum levels of flt-3 ligand fluctuate in an inverserelationship to the degree of bone marrow failure. flt-3 ligand levelsin serum inversely correlate with the colony forming ability in vitro ofbone marrow precursors from patients with aplastic anemia. flt-3 ligandtreatment of mice challenged with syngeneic fibrosarcoma cells has beenshown to result in complete tumor regression and in decreased tumorgrowth rates.

[0321] Antitumor cytokines are especially useful in the methods andcompositions of the invention. According to the invention, an “antitumorcytokine” is a cytokine that can limit the growth or metastasis of tumorcells in vitro or in vivo, or can prolong the survival of atumor-bearing animal, when either admixed with the cells or administeredto the animal. The cytokine can be formulated as a solution in abiologically compatible buffer, e.g. PBS, and admixed with tumor cellsin vitro. The concentration of cytokine may be from about the picomolarrange to about the micromolar range. An antitumor cytokine will, forexample, reduce the growth rate of the cells, e.g. by at least 10%compared to buffer alone, or inhibit metastatic properties of the cells,as may be evidenced by, e.g., increased cell adhesiveness or decreasedability to invade an extracellular matrix substrate, such as anartificial basement membrane. Alternatively, an antitumor cytokine mayinhibit the growth or metastasis of a tumor in vivo, or may prolong thesurvival of a tumor-bearing animal. To evaluate the in vivo antitumoreffects of a cytokine, the cytokine may be formulated in apharmaceutically acceptable carrier and administered, e.g., byintravenous, intratumoral, or intraperitoneal injection. The cytokinemay also be administered in association with cells, such as tumor cellsthat express or are coated with the cytokine.

[0322] Assays for Bioactivity

[0323] According to the invention, it is preferred that a cytokine be“bioactive”, “highly bioactive”, “extremely bioactive”, “nativelybioactive”, or “suprabioactive”. Different levels of bioactivity relateto the ability to induce a change in a leukocyte (other than mereoccupancy of the leukocyte's receptors for the cytokine). According tothe invention, all naturally occuring cytokines are natively bioactive.Many types of assay can demonstrate the bioactivity of a non-naturallyoccurring cytokine. For example, a cytokine may be shown to inducesurvival and/or proliferation of a particular cell type. As anotherexample, a cytokine may change the concentration of an intracellularsecond messenger, such as cAMP, arachidonic acid, calcium ions, orinositol triphosphate. The following are examples of assays forbioactivity:

[0324] Assay 1

[0325] Each well of one or more 60-well Lux microtiter trays is loadedwith 200 FDC-P1 cells in 10 ul Dulbecco's modified Eagle's medium with afinal concentration of 10% newborn calf serum. Cytokine in aconcentration in at most the micromolar range is added to each well in avolume of 5 ul. The tray is incubated for 48 h at 37° C. in 10% CO₂Viable cell counts are performed. The average number of viablecells/well is counted. This assay is useful, for example, foridentifying bioactivity mediated through a murine GM-CSF receptor.

[0326] Assay 2

[0327] Cytokine sample and a recombinant standard identical to anaturally occurring cytokine are each diluted serially in completeRPMI-10 in 96-well flat-bottom microtiter plates. Each dilution isplated in triplicate. CT.4S cells in active log-phase growth arecollected, washed at least twice in complete RPMI-10, and resuspended incomplete RPMI-10 at 1×10⁵ cells/ml. 50 ul of the cell suspension isadded to each well of the plate, which is then incubated for 24 h at 37°C. in 5% CO₂ Tritiated thymidine is added to each well and the plate isincubated for an additional 24 h. The cells are then harvested andtritium incorporation is measured by liquid scintillation counting. Thisassay is useful, for example, for identifying bioactivity mediatedthrough an IL-4 receptor.

[0328] Assay 3 (Colony Formation Assay)

[0329] Agar (4% w/v) is melted in sterile water by boiling 3 min. Theagar is then cooled to 42° C. and added to 42° C. RPMI-15 to a finalconcentration of 0.4%. The solution is maintained at 42° C. Femurs areremoved from young mice using sterile technique. Marrow is collected byflushing the opened ends of the bones with sterile Hank's Balanced SaltSolution (HBSS) using a syringe equipped with a 23G needle. Marrow isplaced in a 15 ml tissue culture tube and vortexed into a cellsuspension. Bone fragments are allowed to settle for 5 min, and thesupernatant suspension is removed. The suspension is adjusted to 7.5×10⁶nucleated cells/ml and diluted 1:100 by adding the 42° C. RPMI with 0.4%agar. 2-fold serial dilutions of cytokine are added to 35 mm tissueculture dishes in a volume <=0.2 ml. Control dishes have no cytokineadded. 1 ml warm cell suspension is added to each dish and the agar isallowed to set at room temperature. The cultures are incubated for 5-7days at 37° C. in 5% CO₂. Colony formation is then evaluated bymicroscopy. The average number of colonies of a given type (or aggregatenumber of colonies of given different types) on the cytokine plates andthe average number on the control plates is counted.. This assay isuseful, for example, for identifying bioactivity mediated through CSFreceptors.

[0330] Assay 4

[0331] Cytokine is diluted serially in RPMI 1640/25 mM HEPES/1% BSA. 25ul of each dilution is plated in triplicate in a multiwell chemotaxischamber bottom. Wells containing medium alone serve as negative controlsand wells containing chemotaxis-inducing naturally occurring cytokineserve as positive controls. A polycarbonate membrane is placed over thechamber bottom and the chamber is assembled. 50 ul of peripheral bloodmononuclear cells at 1.5×10⁶ cells/ml in the RPMI/HEPES/BSA is added toeach of the upper wells of the chamber. The chamber is incubated for 90min at 37° C. in 5% CO₂ The membrane is removed, washed, and stained.Migrated cells in 3-5 random fields of each well are counted bymicroscopy.

[0332] Assay 5

[0333] Naturally-occurring cytokine reference standard is diluted to 2ng/ml in a 17×100 mm tube using supplemented medium. 3 further 5-foldserial dilutions are also prepared. Serial dilutions of cytokine areprepared in 17×100 mm tubes from 2 ng/ml to 20 pg/ml. 50 ul ofPHA-activated human lymphoblasts 4×10⁵ cells/ml in supplemental mediumis added to each well of a 96-well flat-bottom microtiter plate. 50 ulof each dilution of reference standard or cytokine is added totriplicate wells. Negative control wells receive 50 ul of supplementedmedia alone. The plate is incubated for 48 h at 37° C. in 5% CO₂ and thecells are labeled with tritiated thymidine Incorporation is measured byliquid scintillation counting.. This assay is useful, for example, foridentifying bioactivity mediated through an IL-12 receptor.

[0334] Assay 6

[0335] In another assay for bioactivity, an immunocompetent animal isvaccinated with on the order of 10⁴-10⁸ irradiated cytokine-transducedor cytokine-coated tumor cells, and challenged with on the order of10⁴-10⁸ live wild-type tumor cells (in any temporal sequence). Readoutsof the assay are survival, tumor onset, or number of metastases.

[0336] Further examples of cytokine assays can be found, e.g., in:Callard R E et al Assay for human B cell growth and differentiationfactors. in: Clemens M J et al (eds) Lymphokines and Interferons. Apractical Approach, pp. 345-64, IRL Press, Oxford 1987; Coligan J E etal Current protocols in immunology. Grene and Wiley-Interscience, NewYork 1991); Dotsika E N Assays for mediators affecting cellular immunefunctions. Current Opinion in Immunology 2: 932-5 (1989); Feldmann M etal Cytokine assays: role in evaluation of the pathogenesis ofautoimmunity. Immunological Reviews 119: 105-123 (1991); Guiguet M et alMisinterpretation of the biological activity of cytokine-containingpreparations attributable to unrecognized interacting components.Analytical Biochemistry 247(2): 441-442 (1997); Hamblin A S & O'Garra AAssays for interleukins and other related factors. In: Lymphocytes, apractical approach, Klaus G G B (edt), pp. 209-28, IRL Press, Oxford,(1987); Laska E M & Meisner M J Statistical methods and applications ofbioassay. Annu. Rev. Pharmacol. Toxicol. 27: 385-97 (1987); Mosman T R &Fong T A T Specific assays for cytokine production by T cells Journal ofImmunological Methods 116: 151-8 (1989); Newton R C & Uhl J Assaysrelevant to the detection and quantitation of cytokines and theirinhibitors. Modern Methods in Pharmacol. 5: 83-99 (1989); Thorpe R et alDetection and measurement of cytokines. Blood Rev. 6: 133-48 (1992); vanZoelen E J The use of biological assays for detection of polypeptidegrowth factors. Progress in Growth Factor Research 2: 131-52 (1990);Winstanley F P Cytokine bioassay. In: Gallagher G et al (eds) TumorImmunobiology, A practical Approach. Oxford University Press, pp.179-303 (1993); Wadha M et al Quantitative biological assays forindividual cytokines. In: Balkwill F R (edt) Cytokines, A practicalapproach. Oxford University press, pp. 309-330 (1991) According to theinvention, if a non-naturally occurring cytokine gives a readout in abioactivity assay that is at least 10% but not more than 29% (to thenearest 1%) of the readout yielded by an equimolar amount of a naturallyoccurring cytokine (the latter giving a positive result in the assay),then the non-naturally occurring cytokine is “bioactive”. According tothe invention, if a non-naturally occurring cytokine gives a readout ina bioactivity assay that is at least 30% but not more than 49% (to thenearest 1%) of the readout yielded by an equimolar amount of a naturallyoccurring cytokine (the latter giving a positive result in the assay),then the non-naturally occurring cytokine is “highly bioactive”.According to the invention, if a non-naturally occurring cytokine givesa readout in a bioactivity assay that is at least 50% but not more than69% (to the nearest 1%) of the readout yielded by an equimolar amount ofa naturally occurring cytokine (the latter giving a positive result inthe assay), then the non-naturally occurring cytokine is “extremelybioactive”. According to the invention, if a non-naturally occurringcytokine gives a readout in a bioactivity assay that is at least 70% butnot more than 100% (to the nearest 1%) of the readout yielded by anequimolar amount of a naturally occurring cytokine (the latter giving apositive result in the assay), then the non-naturally occurring cytokineis “natively bioactive”. According to the invention, if a non-naturallyoccurring cytokine gives a readout in a bioactivity assay that isgreater than 100% of the readout yielded by an equimolar amount of anaturally occurring cytokine (the latter giving a positive result in theassay), then the non-naturally occurring cytokine is “suprabioactive”.

[0337] Ligands for CD40 Useful According to the Invention

[0338] Nucleotide sequences encoding the CD40 proteins of variousspecies are provided by, e.g., Genbank Accession Nos. Y10507, M83312,and U57745. Human CD40 is a transmembrane glycoprotein with a length of277 amino acids (48 kDa). CD40 is a phosphoprotein and can be expressedas a homodimer. A soluble form of CD40 (28 kDa) has also been described.CD40 protein is expressed on all B-lymphocytes during various stages ofdevelopment, activated T-cells and monocytes, follicular dendriticcells, thymic epithelial cells, and various carcinoma cell lines. It isexpressed on most mature B-cell malignancies and on some early B-cellacute lymphocytic leukemias. CD40 has been demonstrated on the majorityof myeloma cell lines and myeloma cells from patients with plasma celldyscrasia.

[0339] Induction of CD40 mRNA and enhancement of cell surface proteinexpression in primary human monocytes is observed after treatment withGM-CSF , IL3, or IFN-gamma. The human CD40 gene maps to chromosome 20.

[0340] CD40 has been proposed to play a role in the development ofmemory cells. It also plays a role in cell activation, functioning as acompetence factor and progression factor. Crosslinking of the CD40antigen (in combination with cytokines such as IL4 and IL5) leads toB-cell proliferation and induces immunoglobulin class switching from IgMto the synthesis of IgG, IgA, and IgE in the absence of activatedT-cells. CD40 is one of the obligatory signals required for commitmentof naive B-cells to IgA secretion; the mechanism of IgA inductionrequires the cooperation of IL10 and TGF-beta. Soluble CD40 inhibitsT-cell-dependent B-cell proliferation.

[0341] Monoclonal antibodies against CD40 mediate a variety of effectson B-lymphocytes, including induction of intercellular adhesion (viaCD11a/CD18 (LFA-1)), short- and long-term proliferation, differentiationand enhanced tyrosine phosphorylation of proteins. Germinal centercentrocytes are prevented from undergoing cell death by apoptosis byactivation through CD40 and antigen receptors.

[0342] In human resting B-cells expression of CD40 is induced by IL4.Treatment of human B-cells with IL6 leads to the phosphorylation of theintracellular CD40 domain. CD40 does not, however, function as areceptor for IL6. In activated human B-cells the synthesis of IL6 isinduced by treatment of the cells with monoclonal antibodies directedagainst CD40, suggesting that CD40 participates in signal transductionmechanisms dependent on IL6.

[0343] Some limited sequence homologies have been found with receptorsfor Nerve Growth Factor, TNF-alpha and CD27 and it has been assumed thatCD40 may be involved also in modulating the biological activity of theseand other cytokines.

[0344] CD40 has biological functions also in non-immune cells althoughthese are still largely unknown. CD40 ligation has been shown to inducecell death by apotosis in transformed cells of mesenchymal andepithelial origin. In part these processes are mediated through thedeath domain present in the cytoplasmic domain of CD40.

[0345] A particularly useful ligand for CD40 is CD154. CD154 (“CD40ligand”; human protein 29.3 kDa, 261 amino acids) is a member of the TNFfamily of proteins. The human protein shows 82.8% and 77.4% identity atthe CDNA and protein level, respectively, with a similar proteinisolated from murine EL4 thymoma cells. Both proteins are the ligandsfor the CD40 cell surface antigen expressed on resting B-cells. Thehuman gene encoding CD154 maps to chromosome Xq26.3-q27. Nucleotidesequences encoding the native CD40 ligands of various species areprovided by, e.g., Genbank Accession Nos. X67878, X96710, X68550,X65453, Z48469, and L07414. Amino acid sequences of the CD154 moleculesof various species are provided, e.g., by Entrez protein databaseAccession Nos. 1705713, 231718, 560693, 3047129, 116000, 1518170, 38412,109639, 1083014, 38484, and 37270.

[0346] CD154 is naturally synthesized as a transmembrane polypeptide.Nevertheless, a biologically active soluble fragment of human CD154 hasbeen described (Pietravalle et al, 1996, J Biol Chem 271:5965-5967.)Mazzei et al (1995, J Biol Chem 270:7025-7028) identified a biologicallyactive soluble fragment of CD 154 as a homotrimer of polypeptidesconsisting of amino acids Glu 108 through Leu 261 of intacttransmembrane CD154. Graf et al (1995, Eur J Immunol 25:1749) describeanother active fragment consisting of the C-terminal fragment producedby proteolyttic cleavage at Met 113. Aruffo et al disclose soluble formsof CD154 and their use to stimulate B cells in vitro in U.S. Pat. No.5,540,926. In the present invention, particularly useful ligands forCD40 include polypeptides that comprise a sequence as set forth in SEQID NO. 2 of the '926 patent, from amino acid residues 47 to 261. Theseresidues are comprised by the extracellular domain of human CD154.

[0347] Another particularly useful type of ligand for CD40 is anantibody to CD40. Examples of such antibodies include the monoclonalantibodies designated product numbers MCA1143 and MCA1590 of HarlanBioproducts for Science (Indianapolis, Ind.); monoclonal antibodiesdesignated catalog numbers P61640F (produced by clone 14G7), P42374M(produced by clone MAB89), P61046M (produced by clone BL-C4), andP54486M (produced by clone B-B20) of Biodesign International (Kennebunk,Me.); monoclonal antibody designated catalog number 05-422 (produced byclone 626.1) of Upstate Biotechnology (Lake Placid, N.Y.); monoclonalantibody designated catalog number 3601 (produced by clone S2C6) ofMabtech (Nacka, Sweden); monoclonal antibodies designated catalognumbers RDI-CBL486 (produced by clone BB20), RDI-M1691clb (produced byclone CLB-14G7), RDI-mCD40-323 (produced by clone 3/23) of ResearchDiagnostics (Flanders, N.J.); monoclonal antibodies described in Schwabeet al, 1997, Hybridoma 16:217-226; monoclonal antibodies described inBjorck et al, 1994, Immunology 83:430-437; monoclonal antibody G28-5described by Ledbetter et al, 1994, Circ Shock 44:67-72; and monoclonalantibodies described in Buske et al, 1997, Exp Hematol 25:329-337.

[0348] Opsonins Useful According to the Invention

[0349] As defined hereinabove, “opsonin” refers to naturally occurringand non-naturally occurring molecules which bind to both antigens andantigen presenting cells (APCs), such as, for example, phagocyticleukocytes (including monocytes and macrophages), dendritic cells (forexample, Langerhans cells of the skin), B lymphocytes and, in humans,endothelial cells, or molecules which can be processed such that atleast one product of the processing step or steps can bind to bothantigens and antigen presenting cells (APCs), such as, for example,phagocytic leukocytes, dendritic cells, B lymphocytes, and, in humans,endothelial cells.

[0350] Without being bound to any one mechanism of action, it isbelieved that opsonin-enhanced cells provide a beneficial effectaccording to the invention because the opsonin portion acts as a link orcoupling agent between the antigen and the APC to allow more efficientbinding, engulfment, and internalization of the antigen. In addition,the opsonin itself can be internalized with the antigen.“Internalization” refers to the cellular uptake of a molecule such thatit is brought into the cytoplasm or a compartment within the cytoplasmof the cell. Phagocytosis is a process by which a molecule isinternalized by a cell.

[0351] Preferred opsonins are non-rodent opsonins, e.g., primate, e.g.,human, opsonins. Opsonins useful according to the invention bind toreceptors on APCs (e.g., phagocytic leukocytes, e.g., macrophages andother cells of the phagocytic system) such as receptors on cells whichplay a role in innate immunity, as described herein.

[0352] Some sets of opsonins can be regarded as structurally andfunctionally similar. For example, one family comprises fragments ofcomplement components C3 and C4. These two components are highlystructurally homologous, and each possesses an intramolecular thiolesterbond that is broken when a peptide (C3a or C4a respectively) isproteolytically cleaved from the native molecule. Disruption of thethiolester makes available a chemical structure that can form an esterlinkage with an antigen. The moiety of C3 on which this ester bondresides, i.e. the non-C3a moiety, is designated C3b, and C4b is theanalogous product of C4 cleavage. C3b can be further proteolysed byproteins such as factor I to yield fragments such as C3bi and C3d, whichalso remain linked to the antigen via the ester bond.

[0353] There are four structurally unique proteins that are known tofunction as high affinity receptors for biologically active,membrane-bound fragments of C3 and/or C4. CR1 is the major receptor forthe C3b fragment of C3 and C4b fragment of C4. It is expressed onmonocytes and monocyte-derived APCs, among other cell types. CR2 is themajor receptor for the fragment of C3 known as C3d, and is expressed on,e.g., mature B lymphocytes, but not on cells of monocytic lineage. Themajor role of CR2 on B lymphocytes is believed to be directcostimulation of B cells in concert with their cognate antigens.

[0354] CR3 is expressed primarily by neutrophils and monocytes and isalso expressed on FDC, Kupffer cells, and NK cells. CR3 is a C3 fragmentreceptor with a primary specificity for C3bi. CR3 has been proposed asan important organizer of cytoskeletal events necessary for adhesiveinteractions and membrane reorganization during processes such asphagocytosis.

[0355] CR4 is a member of the beta2 integrin family, and its alpha chainis structurally similar to the alpha chain of CR3 and LFA-1. Its primaryphysiologic ligands are believed to be C3d and C3d,g;, however, itsbiologic activities are less well understood than CR3.

[0356] Another example of a family of innate opsonins is the collectins,a group of collagenous C-type lectins that comprises complementcomponent C1q, mannose binding protein, surfactant proteins A and D, andconglutinin. Each molecule comprises a lectin domain that can bind to anantigen, and a collagenous domain that can bind to receptors onphagocytic mononuclear cells, including receptors that are wholly orpartially identical to the C1q receptor (Nepomuceno et al, Immunity 6:11'9-29; Tenner et al, Immunity 3:485-93; Guan et al, J Immunol152:4005-16; Geertsma et al, Am J Physiol 267:L578-84; Miyamura et al,Biochem J 300:237-42; Malhotra et al, J Exp Med 172:955-9; Mathotra etal, Biochem J 293:15-19). Most known collectins comprise multiplepolypeptide chains, in some cases homomeric and in others heteromeric,that are assembled post-translationally, in part by covalentcross-linkage of hydroxyproline and hydroxylysine residues. Collectinsare demonstrated to be opsonins in, for example, Pikaar et al, J InfectDis 172:481-9; Alvarez-Dominguez et al, Infection & Immunity 61:3664-72;Kuhlman et al, J Exp Med 169:1733-45; and Geertsma et al, op cit.

[0357] Among the other innate opsonins useful according to the inventionare C-reactive protein (CRP), alpha-2 macroglobulin, and fibronectin.CRP, a member of the pentraxin family of molecules, binds to receptorson cells of monocytic lineage and has been shown to be an opsonin (Teboand Mortenson, J Immunol 144:231-8; Holzer et al, J Immunol133:1424-30). Alpha-2 macroglobulin, like C3 and C4, comprises aninternal thiolester bond that can be disrupted when the molecule isproteolysed. Such disruption allows covalent binding of the molecule toan antigen, and binding of alpha-2 macroglobulin to an APC can promoteuptake of the conjugate. Fibronectin binds to the alpha 5 beta 1integrin and can also bind to various antigens, allowing it to functionas an opsonin (Cosio, J Lab Clin Med 103:613-9; Czop and Austen, JImmunol 129:2678-81).

[0358] Immunoglobulins (antibodies) can function as opsonins by bindingantigens via their variable regions and APCs via their constant regions.Typically, an immunoglobulin comprises two heavy chains which arecovalently bound to each other and each of which is bound to one lightchain. These heterotetramers can further assemble into higher-orderstructures, such as the pentamers of IgM. Both heavy and light chainvariable regions can contribute to the structure of the antigen bindingsite, whereas the APC binding site is located on the heavy chainconstant region. Recombinant single-chain antibodies have also beendescribed. APC receptors for immunoglobulins include Fc alpha, Fc gamma,Fc epsilon, and Fc mu receptors for IgA, IgG, IgE, and IgM,respectively.

[0359] Opsonins that are naturally expressed by multicellular eukaryoticorganisms are secreted. The latter characteristic distinguishes opsoninsfrom adhesion molecules. A non-naturally occurring molecule containing anaturally occurring APC-binding moiety shall be considered an opsonin ifit contains a moiety through which it can be stably bound or attached toa cell such that the APC-binding moiety is located in the extracellularspace, whether or not the molecule contains an antigen-binding moiety ofa naturally occurring antigen. Moieties through which molecules can bestably bound to a cell include crosslinking moieties, transmembranesequences, and lipid moieties. The preparation of proteins containingthese sequences or moieties is well-known to one of skill in the art.

[0360] An “APC binding moiety of an opsonin” is a sequence or domain ofan opsonin which when included in a chimeric molecule permits binding ofthe chimeric molecule to a receptor that is physiologically expressed onan APC with an affinity at least in the nanomolar range.

[0361] There are a number of examples of opsonin fragments that compriseAPC binding moieties. Such a fragment may be any length so long as itretains an APC binding function; for example, it may be about 40 aminoacids, 100 amino acids, 150 amino acids, 500 amino acids, 800 aminoacids, or even as long as 3000 amino acids. For example, Las Holtet etal, 1994, FEBS Lett 344:242 describe a carboxy-terminal fragment ofhuman α2 m (val1299-ala1451) that binds with high affinity to the α2 mreceptor. Fragments comprising amino acids 1314-1451 of human α2 m andthe corresponding domain of rat α2 m also bind to α2 m receptors, albeitwith 1-2% of the affinities of native α2 m (Van Leuven et al, 1986, JBiol Chem 261:11369; Enghild et al, 1989, Biochemistry 28:1406; Salvesenet al, 1992, FEBS Lett 313:198; Sottrup-Jensen et al, 1986, FEBS Lett205:20).

[0362] Becherer and Lambris, 1988, J Biol Chem 263:14586 describefragments of C3b that bind to CR1, e.g., C3c, fragments of C3 generatedby elastase treatment and comprising the N-terminal of the alpha' chainof C3b, and a synthetic peptide comprising the 42 N-terminal amino acidsof the C3b alpha' chain. A binding sequence in C3 for CR3 has also beendescribed (Wright et al, 1987, PNAS 84:4235).

[0363] “Collagen stalks” of C1q, which are N-terminal fragments obtainedby pepsin digestion, bind to the C1q receptor (Reid, 1981, MethodsEnzymol 80:16; Malhotra et al, 1993, Biochem J 293:15). Malhotra et al,ibid., also provide evidence that an APC binding moiety of conglutininis comprised by its 55 N-terminal amino acids. Ezekowitz (U.S. Pat. No.5,270,199) offers a putative APC binding site in human mannose bindingprotein consisting of nucleotides 370-438 of FIG. 2 in the '199 Patent.In addition, by homology with conglutinin, exon 1 disclosed in the '199Patent may comprise an APC binding moiety.

[0364] An APC binding moiety of IgG comprises the CH2 domain and thelower hinge region, including residues 234-237, as described by Canfieldand Morrison, 1991, J Exp Med 173:1483-91; Lund et al, 1991, J Immunol147:2657-62; and Sarmay et al, 1992, Mol Immunol, 29:633-9.

[0365] Examples of opsonins which can be used in the compositions andmethods of the invention include fibronectin (e.g., Genbank accessionsX02761, K00799, K02273, X82402, X00307, X00739), CRP (e.g., Genbankaccessions X17496, M11880, M11881, M11882), complement components suchas C1q (e.g., Genbank accessions X66295, M22531, X03084, X58861, andSwiss-Prot accessions P02747, P02745), complement fragments such as C3band C3d (e.g., Genbank accessions K02782, K02765), mannose bindingprotein (e.g., Genbank accessions S42292, S42294, X15422), conglutinin(e.g., Genbank accession X71774), alpha-2-macroglobulin (e.g., Genbankaccessions M93264, M11313), and surfactant proteins A (e.g., Genbankaccessions M68519, S48768) and D (e.g., Genbank accessions L40156,X65018, S38981), immunoglobulins, and their homologues among species.TABLE 2 Exemplary Opsonin, APC binding moiety/APC receptor pairs usefulaccording to the invention. Exemplary APC Binding Opsonin MoietyReceptor α-2 macro- Val(1299)-Ala(1451) of α-2m receptor, CD91 globulinhuman α-2m C3b 42 N-terminal amino acids of CR1 the α' chain of humanC3b C3bi C3bi CR2, CR3 C3d C3d CR2, CR4 C1q Collagen stalks Collectinreceptor (Reid, 1981, Methods (Nepomuceno et al., 1997, Enzymol. 80: 16)Immunity 6: 119), CD93 Conglutinin 55 N-terminal amino acids ofCollectin receptor bovine conglutinin MBP 1. Polypeptide encoded by ntCollectin receptor, CD35, 370-438 of FIG. 2, U.S. Pat. CD14 5,270,199 2.Polypeptide encoded by Econ... I of FIG. 2, U.S. Pat. 5,270,199 CRP CRPCRP receptor, FcγRI, FcγRIIa (CD32) Fibronectin Fibronectin α5b1integrin IgG CH2 domain plus lower FcγRI, FcγRII, FcγRIII hingeincluding amino acids 234-237, as described by Lund et al., 1991, J.Immunol. 147: 2657 Surfactant Surfactant Protein A Collectin receptor,CD14 Protein A Surfactant Surfactant Protein D Protein D

[0366] Determination of Opsonicity According to the Invention

[0367] A given naturally occurring opsonin is considered usefulaccording to the invention if it is determined to possess opsonicityaccording to one or more of the following assays, and if it is asecreted molecule.

[0368] Assay 1

[0369] In one assay of opsonicity, as described by O'Rear and Ross inCurrent Protocols in Immunology, 1994, John Wiley & Sons, pp. 13.4.5-9,SRBC bound via a physiologically occurring linkage to the candidateopsonin molecule are obtained. APCs from the species to which thecandidate opsonin is native are suspended at 4×10⁶/ml in ice-cold HBSSwith 1% (w/v) Cohn fraction of BSA. If the candidate opsonin is afragment of C3, the APCs are freshly drawn, uncultivated peripheralblood monocytes. SRBC linked to the candidate opsonin or control SRBC(identical to the former but not linked to the candidate opsonin) aresuspended in the same solution at 2×10⁸/ml. 100 ul of SRBC suspensionand 100 ul of APC suspension are mixed in a 10×75 mm plastic tube. Thetube is rotated at 40 rpm at 37° C. for 2-20 min. A small drop of thesuspension is placed on a slide, covered with a coverslip, and allowedto stand for 5-10 min. Excess fluid can be removed by pressure on thecoverslip, and the coverslip can be sealed to the slide, e.g. with clearnail polish. The slide is examined microscopically, and the percentageof APCs visibly adherent to 4 or more SRBCs is determined. If thepercentage is 50% or greater when there are up to 4×10⁴ candidateopsonin molecules/SRBC', the candidate opsonin can be an opsonin.

[0370] Assay 2 (For protease-activated candidate opsonin)

[0371] Candidate opsonin or radiolabeled Candidate opsonin is treatedwith a 1.5-3 fold molar excess of protease (0.05 M triethanolamine-0. 1M NaCl, pH 8.0, room temperature overnight). In this assay, the proteasecan serve as the antigen or an excess of another antigen can be added.Prior to binding studies, the candidate opsonin-antigen complex isdialyzed against HBSS (4° C.).

[0372] Candidate opsonin -antigen complex binding to monocytes ismeasured by incubating labeled ligand at a concentration up to 1.0 Mwith (1.5-4.0)×10⁶ monocytes in 200 ml volume on ice. Nonspecificbinding of radiolabeled ligands is determined in the presence of a100-fold molar excess labeled candidate opsonin-antigen complex. Theunbound ligand is separated from the cells and cell-bound ligand byrapid vacuum filtration on glass fiber filters. Studies are performed onice to avoid potential complications due to endocytosis. Bindingconstarts and the number of sites per cell are determined by analysisand by nonlinear curve fit. If candidate opsonin-antigen complexaffinity for a monocyte binding site is in at least the nanomolar range,the candidate opsonin is an opsonin.

[0373] Assay 3

[0374] Part I

[0375] To directly evaluate whether candidate opsonin is bound to thesurface of P. carinii, immunoelectron microscopy is performed. P.carinii are isolated from bronchoaveolar lavage (BAL) of moribundinfected rats using TBS with 1 mM calcium to preserve surface-boundcandidate opsonin. Isolated organisms are fixed inperiodate-lysine-paraformaldehyde buffer and embedded in Lowacrylmounting medium (Ted Pella, Inc., Redding, Calif.). Ultrathin sectionsare obtained, blocked with normal goat serum (2%) for 1 h, and incubatedwith either rabbit anti-candidate opsonin or nonimmune rabbit IgG (25mg/ml) overnight. After washing, the sections are subsequently incubatedwith goat and rabbit IgG conjugated to 15 nM colloidal gold (AmershamCorp., Arlington Heights, Ill.). The sections are washed again andexamined on a transmission electron microscope (model 6400:JEOL USA,Inc., Peabody, Mass.).

[0376] Part II

[0377] The attachment of P. carinii to cultured alveolar macrophages inthe presence or absence of antibody to the candidate opsonin or with theaddition of purified candidate is quantified as follows. Adherence of P.carinii to alveolar macrophages is assayed by ⁵¹Cr-labeling theorganisms. P. carinii are isolated from infected rats with TBScontaining 1 mM calcium to prevent loss of surface-bound candidateopsonin. The organisms are radiolabeled by incubation for 8 h at 37° C.in 2 ml of DME containing 20% FCS and 200 mCi of ⁵¹Cr-sodium chromate(New England Nuclear). Normal alveolar macrophages are lavaged fromhealthy rats and plated in tissue culture plates (1×10⁵) cells/well)which are been precoated with normal rat IgG (100 mg/ml×60 min) in orderto ensure firm adherence of the macrophages. After 1 h, the macrophagesare gently washed with HBSS to remove nonadherent cells. >95% ofmacrophages are adherent after this wash. ⁵¹Cr-P. carinii (1×10⁶)containing surface-associated candidate opsonin are added to themacrophages and incubated at 37° C. for an additional hour.Subsequently, nonadherent P. carinii are removed by washing. Themacrophage monolayers containing adherent P. carinii are solubilized in1 N NaOH and quantified. Adherence of P. carinii is defined as:percentage of adherence=(A/A+B)×100, where A=⁵¹Cr-P. carinii associatedwith the monolayer, and B=unattached ⁵¹Cr-P. carinii. To assess theeffect of candidate opsonin on the attachment of P. carinii to alveolarmacrophage lung cells in culture, P. carinii adherence assays areconducted in the presence or absence of a polyclonal rabbit antibodygenerated against the candidate opsonin (100 mg/ml).

[0378] If candidate opsonin binding to P. carinii is apparent in Part Iand if, in Part II, % adherence is diminished in the presence ofanti-candidate opsonin with statistical significance of P<0.05, thecandidate opsonin is an opsonin.

[0379] Assay 4

[0380] Association of bacteria with adherent monocytes is measured asfollows. Endotoxin level in the modified PBS and in all buffers used isbelow 50 pg/ml as determined by the Limulus assay. 5×10³ monocytes inmodified PBS are allowed to adhere to the wells of a Terasaki plate for2 h at 37° C. After nonadherent cells are removed by three washes withPBS, 5×10⁴ FITC-labeled bacteria in 0.5 ml buffer with or without 10-50micrograms/ml of candidate opsonin are added. A-bacteria-to-monocyteratio of 10:1 to 50:1 is used. After 30 min of incubation at 37° C. inthe dark, the nonadherent bacteria are removed by five washes with warmPBS. Assays are performed in quadruplicate; in each well, the number ofbacteria associated with ³100 monocytes is counted under a flourescencemicroscope using ×400 magnification. Results are expressed as the numberof bacteria associated with 100 monocytes. If this number with candidateopsonin can be at least twice that without candidate opsonin, thecandidate opsonin is an opsonin.

[0381] Assay 5

[0382] Part I

[0383] About 1×10⁷ to 6×10⁷ bacteria per ml are incubated (20 min, 0°C.) with 10 mcg/ml of ¹²⁵I-candidate opsonin in a total volume of 0.7ml. of PBS aliquots, 100 ml, of the reaction mixtures are layered over150 ml of an oil cushion (60% dibutyl phthalate, 40% dioctyl phthalate[Eastman Kodak Co., Rochester, N.Y.]), and the mixtures are centrifuged(10,000×g, 60 s, 4° C.). The tip of the tube, containing the cellpellet, is cut with a Mozart razor blade, and the radioactivity iscounted.

[0384] Part II

[0385] APCs are plated in 96-well tissue culture plates (Costar,Cambridge, Mass.) at 2×10⁵ cells per ml the evening before use. 2×10⁶bacteria per well (0.1 ml per well) are added to the culture plates withor without 100 mcg/ml of candidate opsonin. The plates are thencentrifuged at 1,000×g for 7 min. After 15 min at 37° C. to allow theuptake of bacteria, free bacteria are removed by several washes withcold PBS. They are then incubated (45 min, 37° C.) in RPMI 1640 plus anamount of antibiotic that, when present in the culture for 45 min, killsall extracellular bacteria. The end of this incubation period isconsidered time zero. Monolayers are washed three times with Hanks'balanced saline solution, and the same volume of RPMI 1640 (R0) isadded. The cells arc lysed by using several cycles of freezing andthawing. The number (CFU) of viable bacteria per well is determined byquantitative plate counts on blood agar plates (Columbia blood agar;Becton Dickinson, San Jose, Calif.) after 24 h of incubation. Eachresult is given as the mean of three determinations.

[0386] If, in Part I, candidate opsonin-treated bacterial pellet has >75KCPM and this incorporation can be inhibited by unlabeled candidateopsonin, and if in Part II the CFU with candidate opsonin is greaterthan without (P<0.05), the candidate opsonin can be an opsonin.

[0387] Assay 6

[0388] 200 μl of GHBSS (Hanks Balanced Salt Solution) +0.1% of gelatincontaining 10 m mol CaCl₂) containing 10⁷ bacteria is prepared. Thebacteria are then incubated at 4° C. with 20-100 μg/ml of candidateopsonin. Binding assays are done in the presence or absence of acompetitive inhibitor. After incubation for 30 minutes, the bacteria arewashed five times in a GHBSS+10 mmol CaCl₂ at room temperature in amicrofuge at 1,300 g for 3 minutes. Thereafter, a 1:1,000 dilution ofrabbit anti-candidate opsonin antiserum is incubated with the bacteriafor 1 h in PBS +5% FCS and 10 mmol CaCl₂ and then the bacteria arewashed three times in GHBSS+10 mmol CaCl₂ plus 0.05% Tween 20. Bindingof anti-serum to bacteria is detected by a 1:1,000 dilution of goatanti-rabbit IgG conjugated to rhodamine (Fisher Pharmaceuticals,Orangeburg, N.Y.). After incubation, the bacteria are washed five timesin GHBSS+10 mmol CaCl₂ plus 0.05% Tween 20, smeared onto glass slidesand allowed to air dry. Thereafter bacteria are fixed with 100% ice coldmethanol for 5 minutes. Negative controls included the absence ofcandidate opsonin and no first step antibody. Numerous fields oftriplicate assays are examined by fluorescence microscopy.

[0389] Part II Association of Radiolabeled Bacteria with Cells.

[0390] 10⁷ radiolabeled bacteria are resuspended in 200 μl of GHBSS+10mmol CaCl₂ and are incubated with or without candidate opsonin rangingfrom 2 μg/ml to 40 82 g/ml at 4° C. for 30 min. The bacteria are thenwashed three times in GHBSS+10 mmol CaCl₂ for 3 min at room temperaturein a microfuge at 1,300 g, resuspended in 50 μl of GHBSS and added to a1-ml suspension containing on the order of 10⁶ APCs (GHBSS). Thebacteria and APCs are gently rocked at 37° C. for 20 min and thereafterthe unattached bacteria are removed by five washes using differentialcentrifugation at 82 g in a microfuge. Before the last wash, an aliquotfrom each sample is plated on a Labtek slide and cells are adhered for10 min, fixed in methanol, stained with Giemsa, and scored by lightmicroscopy. To score the cells plated on the Labtek slides, at least 400cells are counted. The phagocytic index represented the number ofattached or ingested particles per 100 PMNs. The pellet from abovecontaining cells and radiolabeled bacteria is then lysed in 100 μl PBS+0.5% Triton X-100 and the radioactivity is measured in a scintillationcounter. If, in Part I, specific binding of candidate opsonin tobacteria is evident, and in Part II the specific uptake of bacteria, incpm, is more than three times greater with candidate opsonin thanwithout, the candidate opsonin can be an opsonin.

[0391] Assay 7

[0392] Part I

[0393] To investigate binding to L donovani promastigotes cultures areseeded at 5×10⁵ parasites ml⁻¹. At regular time points up to 9 days, afraction of parasites are counted, washed, and resuspended in 1% BSA,0.5 mM Ca²⁺. 0.05% NaN₃, Tris-buffered saline (TBS), (10 mM Tris-HCl,0.15 M NaCl, pH 8.0) (diluent) to 2×10⁵ ml⁻¹. Fifty microliters of thissuspension are then added to 200-μl microfuge tubes containing 70 μl 5μg/ml radiolabled candidate opsonin (0.12 μCi/μg) in diluent withoutEDTA, which had been layered over 150 μl of a dinonyl phthalate/dibutylphthalate (40:60 v/v) oil mixture. Parasites are incubated for 1 h andcentrifuged through the oil layer, the cell pellet is cut off, andassociated candidate is detected by gamma counting. Each assay isperformed in triplicate. The concentration dependency of candidatebinding to promastigotes is also measured as above, using an activity of0.045 μCi/μg and a twofold dilution series from 60 to 0.015 μg/mlcandidate.

[0394] Part II

[0395] APCs are plated out at 1×10⁶ cells/well on glass coverslips in a24-well tissue culture plate. Cells are incubated in RPMI 1640 (LifeTechnologies) supplemented with 10% PCS, 1 mM glutamine, 200 U/mlpenicillin and 200 μg/ml streptomycin in a humidified incubator at 37°C. After 24 h, nonadherent cells are removed and remaining cells areused after 6 days. Promastigotes are incubated with or without candidateat 30 μg/ml in RPMI 1640 for 1 h and then washed three times beforeadding to the APC cultures at 10⁶/well. Promastigotes are allowed toinfect APCs for 1 h, then cells are washed, fixed with methanol, andGeimsa stained (BDH, Poole, Dorset, U.K.) before counting. Thepercentage of APCs infected and the number of parasites/100 macrophagesis determined from quadruplicate cultures.

[0396] If in Part I the affinity of candidate opsonin for parasites isat least in the nanomolar range and in Part II the number of parasitestaken up/100 APCs is, with candidate opsonin, at least twice thatwithout candidate opsonin, the candidate opsonin can be an opsonin.

[0397] Assay 8

[0398] Part I

[0399] Portions (0.5 ml) of [³⁵S] methionine-labeled culture mediumcontaining 5 percent fetal calf serum and the candidate opsonin areincubated for 30 minutes at room temperature with 0.1 ml or 0.2 ml of a10 percent suspension of a microorganism). The microorganisms tested mayinclude, for example, Salmonella typhimurium, Bacillus subtilis,Staphylococcus aureus, Escherichia coli, and Saccharomyces cerevisiae.Bound proteins are released by boiling in buffer containing 2 percentSDS and 0.1 M dithiothreitol and are analyzed on a 5 percent SDS gel.

[0400] Part II

[0401] Fixed bacteria (0.1 ml; 10 percent by volume; 10¹⁰ organisms permillileter), labeled with [³H]thymidine, are incubated with 0.1 ml ofserum with or without depletion of the candidate opsonin. After beingwashed with PBS, the bacteria are incubated with on the order of 1×10⁷APCs in a final volume of 0.9 ml PBS containing divalent cations. Atintervals 0.2 ml is removed to ice-cold PBS with N-ethyimaleimide (2 mM)to block further endocytosis, and the cells are washed (at about 100 gfor 10 seconds)

[0402] If in Part I a band corresponding to the candidate opsonin isapparent, and if in Part II the CPM after 6-10 min of incubation is atleast three times greater for undepleted samples with serum than withdepleted serum, the candidate opsonin can be an opsonin.

[0403] In lieu of results form Parts I of assays 3, 5, 6, 7, 8, acandidate opsonin that satisfies Part II of an assay can be an opsoninif it can bind to the antigen of the assay with an affinity in at leastthe nanomolar range.

[0404] Assay 9

[0405] SRBC coated with at least 1.2×10⁴ molecules/cell of a fragment ofC3 are prepared as described by O'Rear and Ross in Current Protocols inImmunology, 1994, John Wiley & Sons, pp. 13.4.5-9. 250 ul of monocytesat 2×10⁵ cells/ml of RPMI with 10% fetal calf serum are added to eachwell of an 8-well glass tissue culture plate and incubated at 37° C., 5%CO₂ for 3 h. The monocytes are washed twice with HBSS, and 50 ul of theSRBC at 1.5×10⁸/ml of DVBS²⁺ are added to each well. The plate iscentrifuged at 50 g for 5 min and then incubated at 37° C., 5% CO₂ for 3h. The walls are washed twice with HBSS, fixed with 0.5% glutaraldehyde,and stained with Giemsa stain. If >40% of the monocytes form rosetteswith at least 1 SRBC as determined by light microscopy, the candidatecan be an opsonin.

[0406] Heat Shock Proteins Useful in the Invention

[0407] Heat shock proteins (HSPs) are associated in cells with a broadspectrum of peptides, polypeptides, denatured proteins and antigens withwhich they form complexes. Such HSP-peptide complexes have beendescribed as being useful in vaccines against cancers and infectiousdiseases by Srivastava et al., “Heat shock protein-peptide complexes incancer immunotherapy” in Current Opinion in Immunology (1994),6:728-732; Srivastava, “Peptide-Binding Heat Shock Proteins in theEndoplasmic Reticulum” in Advances in Cancer Research (1993),62:153-177. The HSP-peptide complexes appear to work as vaccines,because they may finction as antigen carrying and presentationmolecules. The development of vaccines using such antigens has beendescribed by Baltz, “Vaccines in the treatment of Cancer” in Am. JHealth-Syst. Pharm. (1995), 52:2574-2585. The antigenicity of heat shockproteins appears to derive not from the heat shock protein itself, butfrom the associated peptides, see Udono et al., “Heat Shock Protein70-associated Peptides Elicit Specific Cancer Immunity” in J. Exp. Med.(1993), 178:1391-1396; Srivastava et al., “Heat shock proteins transferpeptides during antigen processing and CTL priming” in Immunogenetics(1994), 39:93-98; Srivastava, “A Critical Contemplation on the Roles ofHeat Shock Proteins in Transfer of Antigenic Peptides During AntigenPresentation” in Behring Inst. Mitt. (1994), 94:37-47. HSPs appear to bepart of the process by which peptides are transported to the MajorHistocompatibility Complex (MHC) molecules for surface presentation.

[0408] A number of different HSPs have been shown to exhibitimmunogenicity, and are useful in the present invention, including, butnot limited to: gp96, hsp90, hsp100, hsp60, hsp 25 and hsp70, see Udonoet al., supra. and Udono et al., “Comparison of Tumor-SpecificImmunogenicities of Stress-Induced Proteins gp96, hsp90, and hsp 70” inJournal of Immunology (1994), 5398-5403; gp96 and grp94, Li et al.,“Tumor rejection antigen gp96/grp94 is an ATPase: implications forprotein folding and antigen presentation” in The EMBO Journal, Vol. 12,No. 8 (1993), 3143-3151; and gp96, hsp90 and hsp70, Blachere et al.,“Heat Shock Protein Vaccines Against Cancer” in Journal Of Immunotherapy(1993), 14:352-356.

[0409] Heat shock proteins may be purified for use in the presentinvention using a procedure employing DE52 ionexchange chromatographyfollowed by affinity chromatography on ATP-agarose, see Welch et al.,“Rapid Purification of Mammalian 70,000-Dalton Stress Proteins: Affinityof the Proteins for Nucleotides” in Molecular and Cellular Biology (June1985), 1229-1237.

[0410] Adhesion Molecules useful in the Invention

[0411] Adhesion molecules useful in the present invention include anycell-surface protein which is involved in bediating the recognition andadhesion of cell sto their substrate and to other cells. Cellularadhesion molecules can be divided into two primary classes: Ca²⁺dependent (cadherins) and Ca²⁺ independent.

[0412] There are over a dozen different types of Ca²⁺ dependent adhesionmolecules called cadherins. Most cadherins are single-pass transmembraneglycoproteins composed of about 700-750 amino acid residues. The largeextracellular part of the molecule is usually folded into five domains,each containing about 100 amino acid residues. Four of these domainscontain presumptive Ca²⁺ binding sites. Cadherins are often present inthe cell membrane as dimers.

[0413] Cadherens useful in the present invention include, but are notlimited to cadherin E, cadherin N, cadherin BR, cadherin P, cadherin R,cadherin M, cadherin VE, cadherin T&H, cadherin OB, cadherin K, cadherin7, cadherin 8, cadherin KSP, cadherin LI, cadherin 18, fibroblast 1,cadherin, fibroblast 2, cadherin, fibroblast 3, cadherin 23, desmocollin1, desmocollin 2, desmoglein 1, desmoglein 2, desmoglein 3, andprotocadherin 1, 2, 3, 7, 8, and 9.

[0414] The remaining adhesion molecules are Ca²⁺ independent, and, likethe cadherins, may be used as ligands of a cell surface protein on anAPC in the present invention. General classes of adhesion molecules aswell as specific adhesion molecules useful in the present invention areshown below in Table 3. TABLE 3 Selectins L-selectin; E-selectin;P-selectin Integrins α1β1; α2β1; α3β1; α4β1; α5β1; α6β1; α7β1; α8β1;α9β1; αvβ1; αLβ2; αMβ2; αXβ2; αIIbβ3; αvβ3; α6β4; αvβ5; αvβ6; αvβ7;αIELβ7; α11 Immunoglobulin Neural Specific: Adhesion molecule on glia(AMOG); Superfamily L1CAM; Myelin-associated glycoprotein (MAG);Myelin-oligodendrocyte glycoprotein (MOG); NCAM-1 (CD-56); NrCAM; OBCAM;P₀protein; PMP-22protein; Neurofascin; NgCAM Systemic IgCAMS: ALCAM;Basigin (CD147); BL- CAM (CD22); CD44; ICAM-1(CD54); ICAM-3 (CD50);Lymphocyte function antigen-2 (LFA-2); LFA-3 (CD58; MHC molecules;MAdCAM-1; PECAM (CD31); T-cell receptor; VACM-1 Other Adhesion Agrin;CD34; GlyCAM-1; Oligodendrocyte-myelin Molecules glycoprotein (OMGP)

[0415] Defensins Useful in the Invention

[0416] In one embodiment, the portion of the multifunctional moleculewhich is a ligand of a cell surface protein of an APC is a defensin.Defensins are a large family of broad-spectrum antimicrobial peptides,identified originally in leukocytes of rabbits and humans. Defensins,cationic, polar peptides (30-35 aa, 3-4 kDa), are distinguished by aconserved tri-disulfide and largely beta sheet structure. When expressedat the cell surface, defensins have been hypothesized to function as abiocheical barrier against microbial invection by inhibitingcolonization of the epithelium by a wide range of pathogenicmicroorganisms. Defensins useful in the present invention include, butare not limited to human alpha defensins 1-6, human neutrophil peptides1-4, human beta defensin 1 and 2, and rat beta defeinsin 1 and 2.

[0417] Counter-receptors of T cell Co-stimulatory Molecules of theInvention

[0418] In one embodiment of the invention the portion of themultifunctional molecule which is a ligand of a cell surface protein ofan APC is a counter-receptor of a T cell co-stimulatory molecule.Costimulation is defined as a signaling pathway that does more thansimply augment antigen receptor-proximal activation events, but thatintersects with antigen-specific signals synergistically to allowlymphocyte activation. Accordingly, a counter-receptor of aco-stimulatory molecule, useful in the present invention includes, butis not limited to a receptor for one or more of B7-1, B7-2, ICOS:B7h,PD-1:PD-L1/PD-L2, CD48, CD40 ligand, and OX40. Counter-receptors usefulin the present invention include, but are not limited to CD28, CTLA-4,ICOS, PD-1, members of the TNF receptor family, CD40, the major B cellcostimulatory molecule, as well as OX-40, 4-1BB, CD30, and CD27.

[0419] Peptide Linkers

[0420] In one embodiment, the multifunctional molecule is a fusionpolypeptide which comprises one or more amino acids interposed betweenthe first and second parts which bind to cells, e.g. a fusionpolypeptide which comprises a first amino acid sequence which can bindto an antigen bearing target and a second amino acid sequence which canbind to a leukocyte, and which further comprises at least one amino acidinterposed between the first and second parts. The interposed aminoacids may comprise, e.g., a linker sequence intended to lessen sterichindrance or other undesirable interactions between the aforementionedfirst and second parts. For, example, one such type of sequence takesthe form (Gly₃Ser)_(n). Additional useful linkers include, but are notlimited to (Arg—Ala—Arg—Asp—Pro—Arg—Val—Pro—Val—Ala—Thr)₁₋₅ (Xu et al.,1999, Proc. Natl. Acad. Sci. U.S.A. 96: 151-156), (Gly—Ser)_(n)(Shao etal., 2000, Bioconjug. Chem. 11: 822-826), (Thr—Ser—Pro), (Kroon et al.,2000, Eur. J. Biochem. 267: 6740-6752), (Gly—Gly—Gly)_(n)(Kluczyk etal., 2000, Peptides 21: 1411-1420), and (Glu—Lys)_(n)(Klyczyk et al.,2000, supra), wherein n is 1 to 15 (each of the preceding references isalso incorporated herein by reference). In another embodiment, no aminoacids are interposed between the first and second parts.

[0421] Antigens Useful According to the Invention

[0422] 1. Viral Antigens

[0423] Examples of viral antigens include, but are not limited to,retroviral antigens such as retroviral antigens from the humanimmunodeficiency virus (HIV) antigens such as gene products of the gag,pol, and env genes, the Nef protein, reverse transcriptase, and otherHIV components; hepatitis viral antigens such as the S. M, and Lproteins of hepatitis B virus, the pre-S antigen of hepatitis B virus,and other hepatitis, e.g., hepatitis A, B. and C, viral components suchas hepatitis C viral RNA; influenza viral antigens such as hemagglutininand neuraminidase and other influenza viral components; measles viralantigens such as the measles virus fusion protein and other measlesvirus components; rubella viral antigens such as proteins E1 and E2 andother rubella virus components; rotaviral antigens such as VP7sc andother rotaviral components; cytomegaloviral antigens such as envelopeglycoprotein B and other cytomegaloviral antigen components; respiratorysyncytial viral antigens such as the RSV fusion protein, the M2 proteinand other respiratory syncytial viral antigen components; herpes simplexviral antigens such as immediate early proteins, glycoprotein D, andother herpes simplex viral antigen components; varicella zoster viralantigens such as gpI, gpII, and other varicella zoster viral antigencomponents; Japanese encephalitis viral antigens such as proteins E,M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitisviral antigen components; rabies viral antigens such as rabiesglycoprotein, rabies nucleoprotein and other rabies viral antigencomponents. See Fundamental Virology, Second Edition, e's. Fields, B. N.and Knipe, D. M. (Raven Press, New York, 1991) for additional examplesof viral antigens.

[0424] 2. Bacterial Antigens

[0425] Bacterial antigens which can be used in the compositions andmethods of the invention include, but are not limited to, pertussisbacterial antigens such as pertussis toxin, filamentous hemagglutinin,pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterialantigen components; diptheria bacterial antigens such as diptheria toxinor toxoid and other diphtheria bacterial antigen components; tetanusbacterial antigens such as tetanus toxin or toxoid and other tetanusbacterial antigen components; streptococcal bacterial antigens such as Mproteins and other streptococcal bacterial antigen components;gram-negative bacilli bacterial antigens such as lipopolysaccharides andother gram-negative bacterial antigen components; Mycobacteriumtuberculosis bacterial antigens such as mycolic acid, heat shock protein65 (HSP65), the 30 kDa major secreted protein, antigen 85A and othermycobacterial antigen components; Helicobacter pylori bacterial antigencomponents; pneumococcal bacterial antigens such as pneumolysin,pneumococcal capsular polysaccharides and other pneumococcal bacterialantigen components; hemophilus influenza bacterial antigens such ascapsular polysaccharides and other hemophilus influenza bacterialantigen components; anthrax bacterial antigens such as anthraxprotective antigen and other anthrax bacterial antigen components;rickettsiae bacterial antigens such as romps and other rickettsiaebacterial antigen component. Also included with the bacterial antigensdescribed herein are any other bacterial, mycobacterial, mycoplasmal,rickettsial, or chlamydial antigens.

[0426] 3. Fungal Antigens

[0427] Fungal antigens which can be used in the compositions and methodsof the invention include, but are not limited to, candida fungal antigencomponents; histoplasma fungal antigens such as heat shock protein 60(HSP60) and other histoplasma fungal antigen components; cryptococcalfungal antigens such as capsular polysaccharides and other cryptococcalfungal antigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

[0428] 4. Parasite Antigens

[0429] Examples of protozoa and other parasitic antigens include, butare not limited to, plasmodium falciparum antigens such as merozoitesurface antigens, sporozoite surface antigens, circumsporozoiteantigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigenssuch as SAG-1, p30 and other toxoplasma antigen components; schistosomaeantigens such as glutathione-S-transferase, paramyosin, and otherschistosomal antigen components; leishmania major and other leishmaniaeantigens such as gp63, lipophosphoglycan and its associated protein andother leishmanial antigen components; and trypanosoma cruzi antigenssuch as the 75-77kDa antigen, the 56kDa antigen and other trypanosomalantigen components.

[0430] 5. Tumor Antigens.

[0431] Tumor antigens which can be used in the compositions and methodsof the invention include, but are not limited to, telomerase components;multidrug resistance proteins such as P-glycoprotein; MAGE-1, alphafetoprotein, carcinoembryonic antigen, mutant p53, immunoglobulins ofB-cell derived malignancies, fuision polypeptides expressed from genesthat have been juxtaposed by chromosomal translocations, human chorionicgonadotrpin, calcitonin, tyrosinase, papillomavirus antigens,gangliosides or other carbohydrate-containing components of melanoma orother tumor cells. It is contemplated by the invention that antigensfrom any type of tumor cell can be used in the compositions and methodsdescribed herein.

[0432] 6. Antigens Relating to Autoimmunity.

[0433] Antigens involved in autoimmune diseases, allergy, and graftrejection can be used in the compositions and methods of the invention.For example, an antigen involved in any one or more of the followingautoimmune diseases or disorders can be used in the present invention:diabetes mellitus, arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiplesclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmunethyroiditis, dermatitis (including atopic dermatitis and eczematousdermatitis), psoriasis, Sjogren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.Examples of antigens involved in autoimmune disease include glutamicacid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelinproteolipid protein, acetylcholine receptor components, thyroglobulin,and the thyroid stimulating hormone (TSH) receptor. Examples of antigensinvolved in allergy include pollen antigens such as Japanese cedarpollen antigens, ragweed pollen antigens, rye grass pollen antigens,animal derived antigens such as dust mite antigens and feline antigens,histocompatiblity antigens, and penicillin and other therapeutic drugs.Examples of antigens involved in graft rejection include antigeniccomponents of the graft to be transplanted into the graft recipient suchas heart, lung, liver, pancreas, kidney, and neural graft components. Anantigen can also be an altered peptide ligand useful in treating anautoimmune disease.

[0434] Examples of miscellaneous antigens which can be can be used inthe compositions and methods of the invention include endogenoushormones such as luteinizing hormone, follicular stimulating hormone,testosterone, growth hormone, prolactin, and other hormones, drugs ofaddiction such as cocaine and heroin, and idiotypic fragments of antigenreceptors such as Fab-containing portions of an anti-leptin receptorantibody.

[0435] Determination of Binding of a Multifunctional Molecule to aAntigen Bearing Target or APC

[0436] Multiple techniques are known to those of skill in the art fordetecting protein-protein binding. That is, the binding of amultifunctional molecule of the invention to either or both of anantigen bearing target and an APC.

[0437] The association between the multifunctional molecule and anantigen bearing target and/or an APC may be measured for example byFluorescent Resonance Energy Transfer (FRET), wherein one peptide (i.e.,the multifunctional molecule) comprises a fluorescent label moiety, andthe antigen bearing target or APC harbours a second such moiety, andwhere excitation at an appropriate wavelength may result in absorptionof photons by one label, followed by FRET, and emission at a secondwavelength characteristic of the second fluorophore, this emission beingmeasured and corresponding to the amount of antigen bearing target orAPC which is associated with the multifunctional molecule.Alternatively, this association may be measured in one of many otherways which are described more fully below.

[0438] A “fluorescent tag” or “fluorescent group” refers to either afluorophore or a fluorescent protein or fluorescent fragment thereof, orrefers to a fluorescent amino acid such as tryptophan which may beincorporated into a polypeptide. “Fluorescent protein” refers to anyprotein which fluoresces when excited with appropriate electromagneticradiation. This includes proteins whose amino acid sequences are eithernatural or engineered.

[0439] It is additionally preferred that the fluorophores comprisefluorescein and tetramethylrhodamine or another suitable pair. Inanother preferred embodiment, the label comprises two differentfluorescent proteins. It is preferred that fluorescent proteins compriseany protein selected from the group consisting of green fluorescentprotein (GFP), blue fluorescent protein, red fluorescent protein andother engineered forms of GFP.

[0440] Preferably, the polypeptide comprises a cysteine amino acidthrough which the label is attached via a covalent bond. Morepreferably, the label may be attached via a primary amine group such asvia a lysine residue. As will be apparent to a person skilled in theart, it is preferable to avoid using the same chemistry for bothlabelling and immobilising polypeptides of the invention. For example,if the polypeptide is immobilised via cysteine residues, the label isadvantageously attached via lysine residues.

[0441] Preferably, the measuring is performed by fluorescent resonanceenergy transfer (FRET), fluorescence anisotropy or fluorescencecorrelation spectroscopy, or by measuring the binding of a fluorescentpartner polypeptide to an immobilised polypeptide. Techniques forperforming such measurements are well known to those of skill in theart.

[0442] It is preferred that the fluorescence emitting means comprise twodifferent fluorophores, and particularly preferred that the fluorophorescomprise fluorescein and tetramethylrhodamine or another suitable pair.

[0443] As used herein with regard to fluorescent labels for use in FRET,the term “appropriate combination” refers to a choice of reporter labelssuch that the emission wavelength spectrum of one (the “donor” moiety)is within the excitation wavelength spectrum of the other (the“acceptor” moiety).

[0444] Methods of detection without use of label are known in the art.These include detection using surface plasmon resonance to detectchanges in the mass of, for example the multifunctional molecule, whichwould occur if binding of the partner polypeptide increased ordecreased. Such measurements may be made for example using a BIACOREmachine. In this embodiment, the multifunctional molecule is immobilizedon a solid support prior to contacting the molecule with the antigenbearing moiety and/or APC.

[0445] In addition to the above methods, one technique for determing thebinding of a multifunctional molecule of the invention to an antigenbearing moiety and/or and APC involves the use of antibodiesspecifically directed to the multifunctional molecule. Briefly, antigenbearing cells, for example, are incubated with the multifunctionalmolecule of the invention in RPMI 1640, or other suitable buffer, for1-4 hours at 37° C. with shaking. The cells are then washed in PBScontaining 2% FBS, or other cell culture serum. The antigen bearingcells are then incubated with, for example, an FITC labeledanti-multifunctional molecule antibody for 1 hour at 4° C. Afteradditional washing in PBS, the cells are analyzed by flow cytometry,wherein the identification of labeled cells is indicative of the bindingof the multifunctional molecule of the invention to the antigen bearingcell.

[0446] Preparation of a Cell Containing a Recombinant Nucleic AcidAccording to the Invention

[0447] In one embodiment of the present invention, a nucleic acidmolecule encoding a multifunctional molecule of the present invention isintroduced into a host cell capable of expressing the nucleic acidmolecule so as to produce the multifunctional molecule. In oneembodiment, the host cell is permitted to express the nucleic acid exvivo. In an alternate embodiment, the host cell is transfected with thenucleic acid molecule encoding the multifunctional molecule, and thenplaced back into the host animal from which it was obtained, wherein themultifunctional polypeptide molecule is expressed in vivo in the hostanimal.

[0448] Host cells are transfected, as taught herein, via conventionalmethods well-known in the art. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory manuals. Additional examples ofmethods of introducing nucleic acid molecules encoding multifunctionalmolecules are described below. The cells containing the introducednucleic acid molecules encoding, for example, multifunctional moleculeand/or an antigen, can themselves be administered to a subject (as theantigen) according to the methods of the invention, e.g., in a vaccinecomposition.

[0449] A. Introduction of Naked Nucleic Acid into Cells

[0450] 1. Transfection mediated by DEAE-dextran: Naked nucleic acid canbe introduced into cells by forming a mixture of the nucleic acid andDEAE-dextran and incubating the mixture with the cells. Adimethylsulfoxide or chloroquine shock step can be added to increase theamount of nucleic acid uptake. DEAE-dextran transfection is onlyapplicable to in vitro modification of cells and can be used tointroduce nucleic acid transiently into cells but is not preferred forcreating stably transfected cells. Thus, this method can be used forshort term production of a gene product but is not a method of choicefor long-term production of a gene product. Protocols forDEAE-dextran-mediated transfection can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (e's.) Greene PublishingAssociates, (1989), Section 9.2 and in Molecular Cloning: A LaboratoryManual. 2nd Edition. Sambrook et al. Cold Spring Harbor LaboratoryPress, (1989), Sections 16.41-16.46 or other standard laboratorymanuals.

[0451] 2. Electroporation: Naked nucleic acid can also be introducedinto cells by incubating the cells and the nucleic acid together in anappropriate buffer and subjecting the cells to a high-voltage electricpulse. The efficiency with which nucleic acid is introduced into cellsby electroporation is influenced by the strength of the applied field,the length of the electric pulse, the temperature, the conformation andconcentration of the nucleic acid and the ionic composition of themedia. Electroporation can be used to stably (or transiently) transfecta wide variety of cell types and is only applicable to in vitromodification of cells. Protocols for electroporating cells can be foundin Current Protocols in Molecular Biology, Ausubel, F. M. et al. (e's.)Greene Publishing Associates, (1989), Section 9.3 and in MolecularCloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold SpringHarbor Laboratory Press, (1989), Sections 16.54-16.55 or other standardlaboratory manuals.

[0452] 3. Liposome-mediated transfection (“lipofection”): Naked nucleicacid can be introduced into cells by mixing the nucleic acid with aliposome suspension containing cationic lipids. The nucleicacid/liposome complex is then incubated with cells. Liposome mediatedtransfection can be used to stably (or transiently) transfect cells inculture in vitro. Protocols can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (e's.) Greene PublishingAssociates, (1989), Section 9.4 and other standard laboratory manuals.Additionally, gene delivery in vivo has been accomplished usingliposomes. See for example Nicolau et al. (1987) Meth. Enz. 149:157-176;Wang and Huang (1987) Proc. Natl. Acad Sci. SA 84:7851-785S; Brigham etal. (1989) Am. J. Med. Sci. 298:278; and Gould-Fogerite et al. (1989)Gene 84:429-438.

[0453] 4. Direct Injection: Naked nucleic acid can be introduced intocells by directly injecting the nucleic acid into the cells. For an invitro culture of cells, nucleic acid can be introduced bymicroinjection. Since each cell is microinjected individually, thisapproach is very labor intensive when modifying large numbers of cells.However, a situation wherein microinjection is a method of choice is inthe production of transgenic animals (discussed in greater detailbelow). In this situation, the nucleic acid is stably introduced into afertilized oocyte which is then allowed to develop into an animal. Theresultant animal contains cells carrying the nucleic acid introducedinto the oocyte. Direct injection has also been used to introduce nakednucleic acid into cells in vivo (see e.g., Acsadi et al. (1991) Nature332: 815-818; Wolff et al. (1990) Science 247:1465-1468). A deliveryapparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo canbe used. Such an apparatus is commercially available (e.g., fromBioRad).

[0454] 5. Receptor-Mediated DNA Uptake: Naked nucleic acid can also beintroduced into cells by complexing the nucleic acid to a cation, suchas polylysine, which is coupled to a ligand for a cell-surface receptor(see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320). Binding of the nucleic acid-ligand complex to the receptorfacilitates uptake of the nucleic acid by receptor-mediated endocytosis.Receptors to which a nucleic acid-ligand complex have targeted includethe transferrin receptor and the asialoglycoprotein receptor. A nucleicacid-ligand complex linked to adenovirus capsids which naturally disruptendosomes, thereby releasing material into the cytoplasm can be used toavoid degradation of the complex by intracellular lysosomes (see forexample Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850;Cristiano et al. (1993) Proc. Natl. Acad. Sci USA 90:2122-2126).Receptor-mediated nucleic acid uptake can be used to introduce nucleicacid into cells either in vitro or in vivo and, additionally, has theadded feature that nucleic acid can be selectively targeted to aparticular cell type by use of a ligand which binds to a receptorselectively expressed on a target cell of interest.

[0455] Generally, when naked nucleic acid is introduced into cells inculture (e.g., by one of the transfection techniques described above)only a small fraction of cells (about 1 out of 105) typically integratethe transfected nucleic acid into their genomes (i.e., the nucleic acidis maintained in the cell episomally). Thus, in order to identify cellswhich have taken up exogenous nucleic acid, it is advantageous totransfect nucleic acid encoding a selectable marker into the cell alongwith the nucleic acid(s) of interest. Preferred selectable markersinclude those which confer resistance to drugs such as G418, hygromycinand methotrexate. Alternatively, a selectable marker maybe one whichemits a detectable signal upon expression such as green fluorescenprotein or blue fluorescent protein. Selectable markers may beintroduced on the same plasmid as the gene(s) of interest or may beintroduced on a separate plasmid.

[0456] B. Viral-Mediated Gene Transfer

[0457] A preferred approach for introducing nucleic acid encoding a geneproduct into a cell is by use of a viral vector containing nucleic acid,e.g. a cDNA, encoding the gene product. Infection of cells with a viralvector has the advantage that a large proportion of cells receive thenucleic acid, which can obviate the need for selection of cells whichhave received the nucleic acid. Additionally, molecules encoded withinthe viral vector, e.g., by a cDNA contained in the viral vector, areexpressed efficiently in cells which have taken up viral vector nucleicacid and viral vector systems can be used either in vitro or in vivo.

[0458] 1. Retroviruses: Defective retroviruses are well characterizedfor use in gene transfer for gene therapy purposes (for a review seeMiller, A. D. (1990) Blood 76:271). A recombinant retrovirus can beconstructed having a nucleic acid encoding a gene product of interestinserted into the retroviral genome. Additionally, portions of theretroviral genome can be removed to render the retrovirus replicationdefective. The replication defective retrovirus is then packaged intovirions which can be used to infect a target cell through the use of ahelper virus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14 and other standard laboratory manuals. Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are well known tothose skilled in the art. Examples of suitable packaging virus linesinclude φCrip, φCre, _(—)2, and_Am. Retroviruses have been used tointroduce a variety of genes into many different cell types, includingepithelial cells, endothelial cells, lymphocytes, myoblasts,hepatocytes, bone marrow cells, in vitro and/or in vivo (see for exampleEglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988)Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc.Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl.Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci.USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573). Retroviral vectors require target celldivision in order for the retroviral genome (and foreign nucleic acidinserted into it) to be integrated into the host genome to stablyintroduce nucleic acid into the cell. Thus, it may be necessary tostimulate replication of the target cell.

[0459] 2. Adenoviruses: The genome of an adenovirus can be manipulatedsuch that it encodes and expresses a gene product of interest but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle. See for example Berkner et al. (1988) BioTechniques 6:616;Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992)Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Adz, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral nucleic acid (and foreign DNA contained therein)is not integrated into the genome of a host cell but remains episomal,thereby avoiding potential problems that can occur as a result ofinsertional mutagenesis in situations where introduced nucleic acidbecomes integrated into the host genome (e.g., retroviral DNA).Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol.57:267). Most replication-defective adenoviral vectors currently in useare deleted for all or parts of the viral E1 and E3 genes but retain asmuch as 80% of the adenoviral genetic material.

[0460] 3. Adeno-Associated Viruses: Adeno-associated virus (AAV) is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenousnucleic acid is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce nucleic acid into cells. A variety of nucleic acidshave been introduced into different cell types using AAV vectors (seefor example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.268:3781-3790).

[0461] The efficacy of a particular expression vector system and methodof introducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, nucleic acidintroduced into a cell can be detected by a filter hybridizationtechnique (e.g., Southern blotting) and RNA produced by transcription ofintroduced nucleic acid can be detected, for example, by Northernblotting, RNase protection or reverse transcriptase-polymerase chainreaction (RI-PCR). The gene product can be detected by an appropriateassay, for example by immunological detection of a produced protein,such as with a specific antibody, or by a functional assay to detect afunctional activity of the gene product, such as an enzymatic assay. Ifthe gene product of interest to be expressed by a cell is not readilyassayable, an expression system can first be optimized using a reportergene linked to the regulatory elements and vector to be used. Thereporter gene encodes a gene product which is easily detectable and,thus, can be used to evaluate the efficacy of the system. Standardreporter genes used in the art include genes encodingbeta-galactosidase, chloramphenicol acetyl transferase, luciferase andhuman growth hormone.

[0462] Cells Useful According to the Invention

[0463] The invention provides for host cells transfected with nucleicacid constructs encoding a multifunctional molecule of the invention.Host cells useful in the invention include but are not limited to thefollowing.

[0464] A host cell can be any cell which is able to act as a carrier foran antigen according to the invention and thus may be a nucleated cellor a procaryotic cell into which nucleic acid can be artificiallyintroduced. Procaryotic cells useful according to the invention includebacterial cells. Eucaryotic (nucleated) cells useful according to theinvention include cells of a yeast, fungus, cells of a parasite andmammalian cells. Mammalian cells useful according to the inventioninclude but are not limited to fibroblasts, including specializedmesenchymal cells such as a synoviocytes; keratinocytes, epithelialcells, endothelial cells, leukocytes and tumor cells.

[0465] Cell lines useful according to the invention include but are notlimited to B16, CMS-5 fibrosarcoma cells, Cos1 cells and CHO cells,TS/A, Lewis lung carcinoma, RENCA, Dunning rat prostate carcinoma, andcell lines included in the catalogue of the American Type CultureCollection (Manassas, Va.).

[0466] Host cells comprising a nucleic acid molecule encoding amultifunctional molecule of the invention can be prepared frompathogenic cells according to the invention. Pathogenic cells includetumor cells (e.g. B16 cells, CMS-5 fibrosarcoma cells, and cells derivedfrom the tumors included in the section entitled “Tumors for which theInvention is Useful”), and cells derived from pathogenic bacterium,pathogenic fungus, pathogenic virus, pathogenic parasite, or apathogenic arthropod.

[0467] Methods of Detecting Expression From an Artificially IntroducedRecombinant Nucleic Acid Sequence

[0468] The invention provides for methods of detecting a protein (e.g.,a multifunctional molecule) that is expressed from a recombinant nucleicacid molecule that has been artificially introduced into a cell.

[0469] Preparation of Antibodies

[0470] Antibodies specific for a protein useful according to theinvention (e.g., a multifunctional molecule) are useful for proteinpurification, and for the detection of expression of these proteins fromcells into which a recombinant nucleic acid molecule expressing theseproteins has been artificially introduced. By antibody, we includeconstructions using the binding (variable) region of such an antibody,and other antibody modifications. Thus, an antibody useful in theinvention may comprise a whole antibody, an antibody fragment, apolyfunctional antibody aggregate, or in general a substance comprisingone or more specific binding sites from an antibody. The antibodyfragment may be a fragment such as an Fv, Fab or F(ab′)₂ fragment or aderivative thereof, such as a single chain Fv fragment. The antibody orantibody fragment may be non-recombinant, recombinant or humanized. Theantibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and soforth. In addition, an aggregate, polymer, derivative and conjugate ofan immunoglobulin or a fragment thereof can be used where appropriate.

[0471] Although a protein product (or fragment or oligopeptide thereof)of a protein according to the invention (e.g., a multifunctionalmolecule according to the invention) that is useful for the productionof antibodies does not require biological activity, it must beantigenic. Antibodies may be directed to any portion of themultifunctional molecule of the invention. For example, an antibody maybe directed to the lecting portion of the multifunctional molecule or tothe ligand portion of the multifunctional molecule. Peptides used toinduce specific antibodies may have an amino acid sequence consisting ofat least five amino acids and preferably at least 10 amino acids.Preferably, they should be identical to a region of the natural proteinand may contain the entire amino acid sequence of a small, naturallyoccurring molecule. Short stretches of amino acids corresponding to theprotein product of a recombinant nucleic acid encoding a protein usefulaccording to the invention (e.g., a multifunctional molecule accordingto the invention) may be fused with amino acids from another proteinsuch as keyhole limpet hemocyanin or GST, and antibody will be producedagainst the chimeric molecule. Procedures well known in the art can beused for the production of antibodies to the protein products ofrecombinant nucleic acids of the invention.

[0472] For the production of antibodies, various hosts including goats,rabbits, rats, mice etc. . . may be immunized by injection with theprotein products (or any portion, fragment, or oligonucleotide thereofwhich retains immunogenic properties) of the recombinant nucleic acidmolecules encoding proteins useful according to the invention. Dependingon the host species, various adjuvants may be used to increase theimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

[0473] I. Polyclonal Antibodies.

[0474] The antigen protein may be conjugated to a conventional carrierin order to increase its immunogenicity, and an antiserum to thepeptide-carrier conjugate will be raised. Coupling of a peptide to acarrier protein and immunizations may be performed as described (Dymeckiet al., 1992, J. Biol. Chem., 267: 4815). The serum can be titeredagainst protein antigen by ELISA (below) or alternatively by dot or spotblotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317).At the same time, the antiserum may be used in tissue sections preparedas described. A useful serum will react strongly with the appropriatepeptides by ELISA, for example, following the procedures of Green etal., 1982, Cell, 28: 477.

[0475] 2. Monoclonal Antibodies.

[0476] Techniques for preparing monoclonal antibodies are well known,and monoclonal antibodies may be prepared using a candidate antigen(e.g., a mulispecific molecule or a lectin whose level is to be measuredor which is to be either inactivated or affinity-purified, preferablybound to a carrier, as described by Arnheiter et al., 1981, Nature,294;278.

[0477] Monoclonal antibodies are typically obtained from hybridomatissue cultures or from ascites fluid obtained from animals into whichthe hybridoma tissue was introduced.

[0478] Monoclonal antibody-producing hybridomas (or polyclonal sera) canbe screened for antibody binding to the target protein.

[0479] 3. Antibody Detection Methods

[0480] Particularly preferred immunological tests rely on the use ofeither monoclonal or polyclonal antibodies and include enzyme-linkedimmunoassays (ELISA), immunoblotting and immunoprecipitation (seeVoller, 1978, Diagnostic Horizons, 2:1, Microbiological AssociatesQuarterly Publication, Walkersville, Md.; Voller et al., 1978, J. Clin.Pathol., 31: 507; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408;Butler, 1981, Methods Enzymol., 73: 482; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.) or radioimmunoassays (RIA)(Weintraub, B., Principles of radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March 1986, pp.1-5, 46-49 and 68-78). For analysing tissues for the presence or absenceof a protein produced by a recombinant nucleic acid encoding a proteinuseful according to the invention (e.g., multifunctional molecule orportion thereof), immunohistochemistry techniques may be used. It willbe apparent to one skilled in the art that the antibody molecule mayhave to be labelled to facilitate easy detection of a target protein.Techniques for labelling antibody molecules are well known to thoseskilled in the art (see Harlow and Lane, 1989, Antibodies, Cold SpringHarbor Laboratory).

[0481] Determining Whether an Immune Response is Modulated According tothe Invention

[0482] The multifunctional molecules described herein are usefulaccording to the invention to modulate an immune response in amammalian, preferably a human, to an antigen or antigens contained inthe antigen bearing target which is bound to the lectin portion of themultifunctional molecule. In one embodiment, a composition comprising amultifunctional molecule bound to an antigen bearing target isadministered to an animal, preferably a human. The second portion of themultifunctional molecule comprising a ligand for a cell-surface moleculeof an APC targets the composition to antigen presenting cells in theanimal to which the composition has been administered. The antigenbearing target is taken up (i.e., ingested or phagocytosed) by antigenpresenting cells. Alternatively, the multifunctional molecule/antigenbearing target complex is contacted with antigen presenting cells invitro under conditions which allow phagocytosis, wherein the APCs aresubsequently returned to the host organinsm from which they werederived.

[0483] The present invention thus provides a method for modulating animmune response in an mammal comprising administering to the mammal acomposition comprising at least a multifunctional molecule as describedherein. In one embodiment, the composition further comprises an antigenbearing target. In a further embodiment, the composition still furthercomprises an APC.

[0484] An “immune response” refers to stimulation/activation of aselected response involving the immune system, or suppression,elimination, or attenuation of a selected response. In a preferredembodiment, an immune response refers to stimulation/activation of aselected response involving the immune system by about at least 5%, orpreferably between 5 and 50% or more preferably between 50 and 100% orat least 100% or greater, or suppression, elimination, or attenuation ofa selected response by about at least 5%, or preferably between 5 and50% or more preferably between 50 and 100% or at least 100% or greater,as compared to control cells that are not CD 40-ligand enhanced cells.Thus, to modulate an immune response means that the desired response ismore efficient, more rapid, greater in magnitude, and/or more easilyinduced than when an antigen bearing target is contacted with an APC inthe absence of a multifunctional molecule. Different immune responses inthe subject may be modulated differentially, e.g., the cellular immuneresponse may be selectively enhanced while the humoral response may beselectively attenuated, and vice versa.

[0485] The following in vitro and in vivo assays are useful fordetermining whether an immune response is modulated according to theinvention. The assays described in detail below measure stimulation orsuppression of cellular or humoral immune responses to an antigen. Theantigens referred to in the following assays are representative. It willbe apparent to one of skill in the art that an immune response to aselected antigen useful according to the invention may be measured usingone or more of the following assays by adapting the assay to thatantigen.

[0486] I. Detection of Increased Phagocytosis

[0487] The following assay may be used in order to determine whetheropsonin-enhanced cells stimulate phagocytosis by antigen presentingcells.

[0488] Phagocytosis is examined using monocytes that have been adheredat 37° for 30 min in RPMI without added FCS. Sheep erythrocytes areincubated with an opsonin, or its precursor, under conditions such thatthere are no more than 300 of such molecules, on average, are depositedon each erythrocyte. If a precursor is used, coated erythrocytes arethen processed to convert all precursors to the actual candidatemolecule (e.g., See Carlo et al., J. Immunol. 123:523-8(1979)). Freshmonocytes are isolated from the subject, and 5×10⁴-1×10⁵ of these cellssuspended in 0.25-0.5 ml of RPMI medium with 1% BSA. This aliquot isplaced in a tissue culture well and incubated for 30 min at 37° C. Anexcess of coated erythrocytes, suspended at 1.2×10⁸ cells/ml, isoverlain on the monocytes, the plate is centrifuged for 5 min at 50 g,and incubated for 30 min at 37° C. Non-ingested material is removed intwo hypotonic lysis steps using ice-cold lysing buffer before fixing andstaining the adherent cells, and examining the cells under lightmicroscopy. Phagocytosis is quantified by determining the percentage of100 monocytes ingesting one or more target cells, and the total numberof ingested E/100 monocyptes (PI) is recorded. Stimulation ofphagocytosis according to the invention is indicated by a phagocyticindex of equal to or greater than 40.

[0489] Another assay for phagocytosis is as follows: Cells of the murinemacrophage line are harvested and suspended in DMEM-10 at 4×10⁵/ml. 2.0ml of this suspension is aliquoted into individual 3.5 cm cell cultureplates, and the dishes incubated at 37° C. in 5% CO₂ overnight. Targetcells, as well as control cells, are harvested on the same day as themacrophages, washed in PBS, and resuspended 2 min in PKH26 dye (a 2 μMsolution in 1 ml of the supplied diluent) at 5×10⁶ cells/ml. Thefluorescent PKH26 dye emits in the red spectrum when excited, whereasthe FITC label that is used for the phagocytes emits in the greenspectrum. PKH26 is stable in the endosomal/lysosomal compartment ofphagocytes. The dyed target cells are washed 3 times with PBS andcultured overnight to allow leaching of PKH26 out into the medium. Thisminimizes leakage of dye during the assay. The following day the targetcells are harvested, washed 3 times with PBS, and resuspended inserum-free DMEM at 5×10⁵/ml. The phagocytic cells are rinsed vigorouslywith PBS on the culture plates in order to remove serum, and 2 ml oftarget cells is added to each plate After 0, 2, 4, or 8 h, the platesare rinsed 3 times with PBS to remove all non-adhered cells and theremaining cells are incubated with 2 mM EDTA to release them from theplate. The released cells are washed with 1% FBS/PBS, and suspending in100 μl of the same buffer. 2 μg anti-phagocyte (e.g. anti-CR3) antibodyis added and the cells placed on ice for 25 min. The cells are washed 3times with 1% FBS/PBS, resuspended in 100 μl of this solution, andstained with a 1:25 dilution of FITC-conjugated secondary IgG for 25 minon ice. Cells are washed 3 times and resuspended in 500 μl 1% FBS/PBS,then analyzed on a Becton Dickinson FACScan with CellQuest software.

[0490] FL-1 (green) fluorescence is used to gate phagocytes. The FL-2(red) fluorescence of these cells, which reflects internalization ofPKH26-labeled target cells, is then measured. Phagocytosis induced by,e.g., an opsonin is indicated by the difference between mean FL-2fluorescence of macrophages incubated with opsonin-coated versusnon-opsonin-coated target cells. Use of an opsonin will increase meanFL-2 fluorescence by, e.g. at least 10%., or enough to obtain ap valueless than or equal to 0.05 by student t-test.

[0491] II. Amplification of the Immune Response Usually InvolvesProliferation of Particular Subpopulations of Lymphoid Cells that areNormally in the Resting State.

[0492] Proliferative assays have the following applications in clinicalstudies: (1) Assessment of overall immunologic competence of T cells orB cells as manifested in their ability to respond to polyclonalproliferation signals such as mitogens or anti-CD3 antibodies. Defectsin the proliferation may be indicative of fundamental cellularimmunologic defect. Low proliferation is often found as a nonspecificsecondary effect of chronic disease. (2) Assessment of an individual'sresponse to specific antigens, where low responses are indicative ofgeneral or specific immunologic defect. (3) Determination of MHCcompatibility by the mixed lymphocyte reaction (MLR).

[0493] In addition, proliferative assays are useful for estimatinglymphokine production, investigating signal transduction, and assessinggrowth factor requirements (e.g., lymphokines) for T or B cells. Theprocedure outlined here measures incorporation of [³H]thymidine intoDNA, which usually correlates well with cell growth as measured bychanges in cell number. However, when the activation stimulus is toxic,as with chemical activators such as ionomycin plus phorbol myristateacetate (PMA), the burst of new DNA synthesis following activation maynot be accompanied with a net increase in viable cells, and, in fact, adecline in cell number may be observed. In this instance, [³H]thymidineincorporation in DNA is more indicative of initial cell stimulation thanestimation of cell number. In addition, [³H]thymidine incorporationprovides information on cell populations, not on individual cells.Alternate methods, such as flow cytometry may be used for studiesrequiring that type of information.

[0494] Assay For Antigen-Induced T Cell Proliferation

[0495] This protocol is designed to test the proliferation of T cells inresponse to a specific antigen—tetanus toxoid. It can be modified totest T cell proliferation in response to any protein or polysaccharideantigen. Materials: (T cell suspension, autologous antigen-presentingcell suspension (non-T cells), Tetanus toxoid solution (Connaught orState Laboratory Institute of Massachusetts)). (1) Count T cells andadjust to 1×10⁶ cells/ml with complete RPMI-10 AB. (2) Treatantigen-presenting cells with mitomycin C (or irradiate with 2500 rad)as in step 2 of one-way MLR protocol. Adjust concentration ofantigen-presenting cells to 2×10⁵ cells/ml. Antigen-presenting cells canconsist of autologous non-T cells or autologous monocytes/macrophages.(3) Add 100 ul T cell suspension and 50 ul antigen-presenting cellpopulation to wells; mix just before dispensing. (4) Add 50 ul tetanustoxoid solution to give final concentrations of 0, 1, 5, 10, and 20ug/ml. Prepare three wells for each dilution. (5) Incubate 6 days in ahumidified 37° C., 5% CO₂ incubator. (6) Pulse with [³H]thymidine andharvest as described in support protocol.

[0496] Assay For Lymphokine-Dependent Cell Proliferation

[0497] This protocol assays the lymphokine-dependent proliferation of alymphocyte population, in this case, the IL-4 dependent proliferation ofB cells. Materials: (Tonsil B cell suspension, Anti-IgM cross-linked toSepharose beads (Bio-Rad), 10,000 U/ml human rIL-4 (Genzyme) in completeRPMI-10). (1) Count tonsil B cells and adjust concentration to 1×10⁶cells/ml with complete RPMI-10. (2) Dispense 100 ul of tonsil B cellsinto each well. Prepare three wells for each experimental condition. (3)Dilute 10,000 U/ml rIL-4 solution 1:10, 1:100, and 1:1000. Add 20 ul ofthe stock or dilution to appropriate wells to yield 1000 U/ml, 100 U/ml,10 U/ml, and 1 U/ml. Include a control well with no rIL-4. (4) Pipetanti-IgM beads into appropriate wells.

[0498] Determine the optimal concentration of beads with pilotexperiments. It is best to include several concentrations of beads ineach experiment to “bracket” the optimal dose. Prepare wells with tonsilB cells and IL-4 dilutions alone, anti-IgM beads alone, culture mediumalone, and all the combinations of IL-4 and anti-IgM bead dilutions. (5)Increase the volume of each well to 200 ul with complete RPMI-10 asnecessary. (6) Culture 5 days in a humidified 37° C., 5% CO₂ incubator.(7) Pulse with [³H]thymidine and harvest as described in supportprotocol.

[0499] [³H]Thymidine Pulse And Harvest Of Cell Cultures

[0500] This protocol is used in conjunction with the preceding protocolsto complete the [³H] thymidine incorporation assay. (1) Add 20 ul of 50uCi/ml [³H]thymidine to each culture (1.0 uCi) at a fixed time beforeterminating the culture (usually 6 or 18 hr). (2) Harvest cell culturesusing an automated multiwell harvester that aspirates cells, lysescells, and transfers DNA onto filter paper, while allowingunincorporated [³H]thymidine to wash out. Fill and aspirate each row ofthe microtiter plate ten times to ensure complete cell transfer andcomplete removal of unincorporated thymidine. Wash each filter stripwith 100% ethanol to facilitate drying. Transfer to scintillation vials.For semiautomated harvester, transfer filter dots for each well intoscintillation counting vials. For manual transfer, dry filters underlamp and transfer to scintillation vial with forceps. Add scintillationfluid to each vial. (3) Count samples in scintillation counter untilstandard deviation is less than 2%. Calculate mean cpm for backgroundcultures and for each experimental condition. There should be less than20% variation in replicate cultures.

[0501] III. Induction And Measurement Of In Vitro Antibody Responses

[0502] The capacity of the human immune system to mount an antibodyresponse following in vivo immunization with a protein or polysaccharideantigen is a revealing indication of the overall integrity of both the Band T cell arms of the immune system. As such, in vivo immunizationfollowed by measurement of the antibody response is an appropriate testof immune function in the various acquired and congenitalimmunodeficiencies and in a host of other conditions affecting theimmune system. The following procedures are for in vivo immunization andfor the measurement of the subsequent immune response using an ELISAtechnique.

[0503] Immuno-Enzymetric Assay For Cytokines Using NIP- And HRPO-LabeledAntibodies

[0504] This protocol describes an immunonoenzymetric assay for cytokinesusing a heterogeneous, noncompetitive immunoassay reaction in which thecytokine is immobilized by a coating antibody bound to a microtiterplate. Unbound material is washed free, and detection is carried outusing a different anti-cytokine antibody labeled with the haptennitroiodophenyl (NIP). This is in turn detected by a horseradishperoxidase (HRPO) conjugate of an anti-NIP antibody, which is revealedwith the chromogenic substrate ABTS. In this noncompetitive immunoassay,the immunoassay signal (A₄₀₅) increases as a direct function of theamount of cytokine present in the sample. Antibodies are prepared asdescribed in Current Protocols in Immunology, 1995, 6.20.2-6.20.10.

[0505] Coat assay plate. (1) Using a multichannel pipettor, transfer 100ul of an appropriate dilution of coating antibody into all wells of theassay plate that are to be used. (2) Seal plates with microtiter platesealer or Parafilm and incubate 2 hr. At 37° C. Prepare samples andstandards in preparation plate. (3) Dilute each sample (or aliquot ofconditioned medium) to be assayed with an equal volume of immunoassaydiluent. (4) Pipet less than or equal to 1 ml of each diluted sample tobe assayed into the upper chamber of a separate Spin-X microfiltrationdevice. Microcentifuge 5 min. At 10,000 rpm and save the filtrates thatcollect in the lower chambers. (5) Add 65 ul of each diluted sample tothe appropriate well of a preparation plate (i.e., a separate 96-wellmicrotiter plate). (6) Thaw an aliquot of cytokine standard at roomtemperature and make sure that it is well mixed. Pipet 130 ul into thewell of the preparation plate representing the highest concentration onthe standard curve. Transfer 65 ul from this well into the next, thencontinue performing serial 1:1 dilutions in immunoassay diluent so that65 ul of each concentration represented on the standard curve is placedin appropriate well of the preparation plate. (7) Thaw an aliquot ofcalibrator at room temperature (if used). Dilute with an equal volume ofimmunoassay diluent, then pipet 65 ul of diluted calibrator intoappropriate well or wells of preparation plate.

[0506] Incubate with coating antibody. (8) Remove coated assay platefrom incubator. Dip in 2-liter beaker filled with 1× wash buffer, theninvert over sink and flick to remove liquid. Repeat two more times, thenbang dry on paper towel. (9) Transfer 50 ul of solution from each wellof preparation plate to corresponding well of the assay plate usingmultichannel pipettor. (10) Seal plate with microtiter plate sealer orParafilm and incubate 2 hr. at room temperature.

[0507] Incubate with detecting antibody. (11) Dilute NIP-labeleddetecting antibody specific to cytokine of interest to 1 ug/ml indetecting buffer. (12) Wash assay plate as in step 8. (13) Add 75 uldiluted detecting antibody from step 11 to all wells of assay plate,including unused outer walls. (14) Reseal plate with microtiter platesealer or Parafilm and incubate 1 hr. at room temperature.

[0508] Incubate with HRPO-conjugated anti-NIP antibody. (15) DiluteHRPO-conjugated anti-NIP Mab 1:3000 in detecting buffer. (16) Wash assayplate as in step 8. (17) Add 75 ul of diluted HRPO-labeled anti-NIPantibody from step 15 to all wells of assay plate. (18) Reseal platewith microtiter plate sealer or Parafilm and incubate 1 hr. at roomtemperature.

[0509] Incubate with chromogenic substrate. (19) Wash assay plate as instep 8. (20) Add 100 ul ABTS substrate working solutions to all wells ofassay plate. Cover plate and incubate at room temperature until colordevelopment reaches desired level (generally until A₄₀₅ for wellscontaining the highest concentration of standard is between 1.5 and 2).This protocol usually produces an assay that can be read after 30 to 60min.

[0510] Read plate and analyze data. (21) Using microtiter plate readerwith computer interface, measure absorbance in all wells at 405 nm insingle-wavelength mode or at 405 and 650 nm in dual-wavelength mode.(22) Fit standard data to a curve described by a first-degree (linear),second degree (quadratic), or four-parameter (nonlinear) mathematicalfunction using curve-fitting software. (23) Interpolate absorbance datafrom unknown cytokine samples to fitted standard curve, and calculatecytokine concentrations.

[0511] IV. Induction of an in vivo Antibody Response Provides anApproach to the Evaluation of the Overall Integrity of the ImmuneSystem.

[0512] In the protocols presented here, diptheria and tetanus toxoidsare used as representative protein antigens and pneumococcalpolysaccharides are used as representative polysaccharide antigensbecause of their safety and availability. It should be noted, however,that the responses elicited by these antigens are likely to be secondaryresponses because of past vaccination or natural exposure. To obtain aprimary response, an unusual antigen such as keyhole limpet hemocyaninshould be used.

[0513] When antigens are administered by the intramuscular orsubcutaneous route, as they are here, a “systemic” immune response isinduced and measurement of circulating antibody is most appropriate. Itis, however, sometimes of interest to evaluate “local” or mucosal immuneresponses. In this case, the antigen is given either intranasally tostimulate respiratory lymphoid tissue or orally to stimulategastrointestinal lymphoid tissue and bronchial washings or intestinalfluids, rather than blood, is assayed for antibody content; in addition,antigens are used that are more appropriate for stimulation of thelocal/mucosal response (i.e., influenza virus antigen for respiratoryresponses and cholera toxin for gastrointestinal responses).

[0514] In assaying the in vivo antibody response, it is important todetermine responses to both protein and polysaccharide antigens becausethese antigens stimulate different components of the immune system. Inthis regard, the major antibody response to protein antigen is composedof IgG1 and IgG3 subclass antibodies, whereas the major antibodyresponse to polysaccharide antigen is composed of IgG2 subclassantibody.

[0515] A variety of immunoassay techniques have been used to measureantibody responses in materials obtained after in vivo immunization. Ofthese, the ELISA assay is perhaps the most useful because it yields astable, easily measurable, reproducible, and safe readout.

[0516] Induction Of In Vivo Antibody Responses To Protein/PolysaccharideAntigens In this protocol antigens are administered by the intramuscularor subcutaneous route and serum is collected for measurement ofresponses. (1) Draw preimmunized blood sample, allow blood to clot, andseparate serum from clot by centrifugation. Store serum at −20° C. to−70° C. in appropriately labeled plastic tubes. (2) Inject 0.5 ml oftoxoid mixture into an appropriately prepared intramuscular site(deltoid or thigh), taking care not to inject material intravenously.(3) Inject 0.5 ml polyvalent pneumococcal vaccine into an appropriatelyprepared subcutaneous site, taking care not to inject materialintravenously. (4) Draw post-immunization blood samples at desiredintervals, usually at 1, 2, and 3 weeks. Separate serum and store at−20° C. to −70° C. (5) After all serum samples are collected, assaysamples for presence of antibodies using ELISA.

[0517] The ELISA offers a rapid, sensitive, reproducible, nonradioactivemethod for measuring in vivo antibody responses to a variety ofantigens, including protein and polysaccharide antigens in sera obtainedfrom individuals vaccinated with tetanus and diphtheria boosters and thepolyvalent pneumococcal polysaccharide vaccine. Assays specific fortetanus, diphtheria and the pneumococcal polysaccharide types I, II, andIII are detailed in Current Protocols in Immunology, 1995, Vols. 6 and7.

[0518] Assay Using Tumor Rejection

[0519] In another assay for immunomodulation, an immunocompent animal isvaccinated with on the order of 10⁴-10⁸ irradiated cytokine-coated tumorcells, and challenged with on the order of 10⁴-10⁸ live wild-type tumorcells (in any temporal sequence). If survival or tumor onset in theseanimals differs from that of animal vaccinated, using identicalparameters, with irradiated non-cytokine coated cells instead ofopsonin-enhanced cells, immunomodulation has occurred. For example, ifat least 10% of the animals in the test group survive 100% longer thanmean survival in the control group, the test is positive. As anotherexample, onset of tumors in 20% of the test animals might be 50% laterthan mean onset in the control animals.

[0520] Dosage and Administration

[0521] The invention encompasses methods of modulating an immuneresponse in a mammal to a selected antigen, the method comprisingadministering to a mammal a therapeutic amount of a compositioncomprising a multifunctional molecule as described herein, or acomposition comprising a multifunctional molecule of the invention andan antigen bearing target, or administering a composition comprising atherapeutic amount of APCs which have been contacted with amultifunctional molecule and antigen bearing target in vitro.

[0522] Compositions described herein may be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in or suspension in, liquid prior to infection can also beprepared. The preparation can also be emulsified, or encapsulated inliposomes. The active immunogenic ingredients are often mixed withcarriers which are pharmaceutically acceptable and compatible with theactive ingredient. The term “pharmaceutically acceptable carrier” refersto a carrier that does not cause an allergic reaction or other untowardeffect in subjects to whom it is administered. As used herein, a“pharmaceutically acceptable carrier” does not include culture medium,or any solution containing about 0.2-2% serum or greater. Suitablepharmaceutically acceptable carriers include, for example, one or moreof water, saline, phosphate buffered saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance the effectiveness of the vaccine. Examples of adjuvantswhich may be effective include but are not limited to: aluminumhydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-alanine-2-1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(COP) 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosporyl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. Other examples of adjuvants include DDA(dimethyldioctadecylammonium bromide), Freund's complete and incompleteadjuvants and QuilA. In addition, immune modulating substances such aslymphokines (e.g., IFN-, IL-2 and IL-12) or synthetic IFN-inducers suchas poly I:C can be used in combination with adjuvants described herein.

[0523] Compositions of the invention can be administered parenterally,by injection, for example, either subcutaneously or intramuscularly.Additional formulations which are suitable for other modes ofadministration include suppositories, and in some cases, oralformulations or formulations suitable for distribution as aerosols. Inthe case of the oral formulations, the manipulation of T-cell subsetsemploying adjuvants, antigen packaging, or the addition of individualcytokines to various formulations can result in improved oral vaccineswith optimized immune responses. For suppositories, traditional bindersand carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of active ingredient, preferably 25-70%.

[0524] The compositions of the invention can be formulated into thevaccine compositions as neutral or salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with free aminogroups of the peptide) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or with organic acidssuch as acetic, oxalic, tartaric, maleic, and the like. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

[0525] Any cellular component of such vaccine compositions can, inpreparation for inclusion in such compositions, be subjected totreatments which involve attenuation or inactivation of the cells of thevaccine, including, for example, exposure to ionizing radiation, whichcan inhibit cell division, antiproliferative agents such ascyclophosphamide, cytochalasin D, or colchicine, or killing with orwithout fixation.

[0526] The compositions, including antigen bearing targets and APCs areadministered in a manner compatible with the dosage formulation, and insuch amount as will be prophylactically and/or therapeuticallyeffective. The quantity to be administered depends on the subject to betreated, including, e.g., capacity of the subject's immune system tosynthesize antibodies, and the degree of protection desired. Suitabledose ranges are on the order of several hundred micrograms activeingredient per vaccination with a preferred range from about 0.1 μg to1000 μg, such as in the range from about 1 μg to 300 μg, and preferablyin the range from about 10 μg to 50 μg. Suitable regiments for initialadministration and booster shots are also variable but are typified byan initial administration followed by subsequent inoculations or otheradministrations. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and may bepeculiar to each subject. It will be apparent to those of skill in theart that the therapeutically effective amount of cells of this inventionwill depend, inter alia, upon the administration schedule, the unit doseof antigen administered, whether the cells are administered incombination with other therapeutic agents, the immune status and healthof the recipient, and the therapeutic activity of the particularcomposition.

[0527] The compositions can be given in a single dose schedule, orpreferably in a multiple dose schedule. A multiple dose schedule is onein which a primary course of vaccination can include 1-10 separatedoses, followed by other doses given at subsequent time intervalsrequired to maintain and or reinforce the immune response, for example,at 1-4 months for a second dose, and if needed, a subsequent dose(s)after several months. Periodic boosters at intervals of 1-5 years,usually 3 years, are preferable to maintain the desired levels ofprotective immunity. The course of the immunization can be followed byin vitro proliferation assays of peripheral blood lymphocytes (PBLs)co-cultured with ESAT6 or ST-CF, and by measuring the levels ofIFN-released from the primed lymphocytes. The assays can be performedusing conventional labels, such as radionucleotides, enzymes,fluorescent labels and the like. These techniques are known to oneskilled in the art and can be found in U.S. Pat. Nos. 3,791,932,4,174,384 and 3,949,064, which are hereby incorporated by reference.

[0528] Tumors for Which the Invention is Applicable

[0529] The invention contemplates treatment of tumors including but notlimited to the following:

[0530] Melanomas, squamous cell tumors, basal cell carcinomas,astrocytomas, gliomas, glioblastoma multiforme, meningiomas,ependymomas, schwannomas, neuroblastomas, retinoblastomas, meningiomas,glomus tumors, sarcomas, including, e.g., osteosarcomas, Ewing'ssarcomas, chondrosarcomas, myosarcomas, synovial cell sarcomas,fibrosarcomas, spindle cell tumors, angiosarcomas, primitiveneuroectodermal cell tumors, and Kaposi's sarcomas, lymphomas, acute andchronic leukemias, tumors of the head and neck, nasopharyngealcarcinomas, carcinomas of the pharynx, laryngeal carcinomas, carcinomasof the thyroid, carcinomas of the parathyroids, thymomas, esophagealcarcinomas, gastric carcinomas, tumors of the small bowel, carcinomas ofthe colon and rectum, mesotheliomas, lung carcinomas, includingadenocarcinomas, squamous cell carcinomas, bronchoalveolar carcinomas,and small cell tumors, pancreatic carcinomas, islet cell and non-isletcell tumors, carcinomas of the breast, cardiac myxomas, pituitarytumors, carcinoid tumors, hepatomas, cholangiocarcinomas,hepatoblastomas, renal cell carcinomas, nephroblastomas, Wilms' tumors,adrenal carcinomas, pheochromocytomas, germ cell tumors,choriocarcinomas, ovarian carcinomas, testicular tumors, seminomas,endometrial tumors, carcinomas of the prostate, carcinomas of theseminal vesicles, vaginal tumors, carcinomas of the penis, hydatiformmoles, carcinomas of the gall bladder, and carcinomas of the urinarybladder.

[0531] Transgenic Animals According to the Invention

[0532] A nucleic acid molecule encoding a multifunctional molecule asdescribed herein can be used to produce nonhuman transgenic animals, andcells of such transgenic animals can be isolated and used in a vaccineformulation in animal or human vaccination.

[0533] For example, in one embodiment, a nucleic acid molecule isintroduced into a fertilized oocyte or an embryonic stem cell. Suchcells can then be used to create non-human transgenic animals in whichexogenous nucleic acid molecules encoding the polypeptides of theinvention have been introduced into their genome or homologousrecombinant animals in which endogenous nucleic acid molecules have beenaltered. Such animals are useful for studying the function and/oractivity of the molecules of the invention and for identifying and/orevaluating modulators of the activity of the molecules of the invention.As used herein, a “transgenic animal” is a non-human animal, prefersmammal, more preferably a mouse, in which one or more of the cells ofthe animal includes a transgene. A transgene is exogenous nucleic acidwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal,thereby directing the expression of an encoded gene product in one ormore cell types or tissues of the transgenic animal.

[0534] A transgenic animal of the invention can be created byintroducing nucleic acid molecules encoding the polypeptides describedherein (i.e., a multifunctional molecule) into the male pronuclei of afertilized oocyte, e.g., by microinjection, and allowing the oocyte todevelop in a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of a polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the nucleic acid molecule of the invention, e.g., thetransgene in its genome and/or expression of the transgene mRNA intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene encoding polypeptides of theinvention can further be bred to other transgenic animals carrying othertransgenes.

[0535] The invention is further illustrated by the followingexemplifications which should not be construed as being furtherlimiting.

EXAMPLES Example 1

[0536] Cloning of a Murine GM-CSF Fused to the S. cerevesiae Gas1 GPIModification Signal Sequence

[0537] The starting point for producing a yeast expression vector wasthe pUC19-GM-CSF-mammalian GPI signal sequence plasmid(pUC19-GM-CSF-GPI). This plasmid encodes murine GM-CSF (upstream) fusedin-frame to the human Thy-1 GPI modification signal sequence(downstream). The following two oligonucleotides were purchased fromMidland Certified Reagent Company (Midland, Tex.): GTX-55′pAATTCCGCGCCGGCACAGTGCTCAGAGACAAACTGGTCAAGTGTGAGGGCATCAGCCTGCTGGCTCAGAACACCTCGTGGCTGCTGCTGCTCCTGCTGTCCCTCTCCCTCCTCCAGGCCACGGATTTCATGTCCCTGTGACTGGGTA C3′

[0538] GTX-5 Comprises:

[0539] a. Sequences at the 5′ end suitable for ligating to an EcoRI site(bases 1-5)

[0540] b. An NgoM1 site for creating an in-frame chimeric codingsequence (bases 9-14)

[0541] c. The coding sequence for the GPI modification sequence of humanThy-1 (Genbank Accession No. M11749) (bases 15-137)

[0542] d. A termination codon (bases 138-140)

[0543] e. Sequences at the 3′ end for ligating to a KpnI site (bases144-148) GTX-6 5′pCCAGTCACAGGGACATGAAATCCGTGGCCTGGAGGAGGGAGAGGGACAGCAGGAGCAGCAGCAGCCACGAGGTGTTCTGAGCCAGCAGGCTGATGCCCTCACACTTGACCAGTTTGTCTCTGAGCACTGTGCCGGCGCGG3′

[0544] This oligonucleotide is complementary to GTX-5, except forstaggered ends.

[0545] GTX-5 and GTX-6 were dissolved in individual tubes in sterilewater at a final concentration of 1 microgram/lambda. GTX-5 and GTX-6were mixed at a final concentration of 100 ng/lambda and allowed toanneal for 60 minutes at room temperature.

[0546] The GTX-5:GTX-6 double stranded oligonucleotide was then clonedinto the plasmid pUC19. Four micrograms of pUC19 DNA was digested withEcoRI and KpnI. After electrophoresis, the linear DNA was purified froma 0.7% agarose gel using a Qiagen (Santa Clarita, Calif.) gelpurification kit according to instructions provided by the manufacturer.100 ng of the GTX-5:GTX-6 oligonucleotide was ligated to 200 ng of theEcoRI-KpnI digested pUC19 in a final volume of 20 microliters at roomtemperature for 60 minutes.

[0547] The plasmid was transformed into competent AG-1 cells, which werepurchased from Stratagene. Transformed E. coli were inoculated ontoLB-amp plates. Bacterial colonies grown on LB plates containingampicillin (100 micrograms/ml) were picked and inoculated into one ml ofLB with amp and grown overnight at 37° with shaking.

[0548] Plasmid DNA was isolated using a standard alkaline lysis miniprepprotocol and DNA was digested with EcoRI and KpnI. DNA waselectrophoresed on 1.6% agarose gels stained with ethidium bromide, andcolonies containing an EcoRI-KpnI fragment of approximately 148 bp werethus identified. Positive colonies were inoculated into 100 ml of LBwith ampicillin and grown overnight. Plasmid DNA was again purifiedusing kits purchased from Qiagen.

[0549] The nucleotide sequence of a Thy-GPI positive clone, designatedpUC-GPI 21, was sequenced, confirming its identity.

[0550] The GM-CSF coding sequence was amplified by PCR from a mouse lungcDNA library purchased from Clontech. PCR was performed for 35 cyclesusing pfu polymerase and the following primers: Upstream5′CCGAATTCATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTAC3′ Downstream5′CAGCCGGCTTTTTGGACTGGTTTTTTGCATTCAAAGGGGATATCAGTCAG3′

[0551] PCR parameters were denaturation at 900 for 1 minute, annealingat 60° for 1 minute, and extension at 72° for 1 minute

[0552] The GM-CSF chain PCR product was purified after electrophoresisthrough a 1% agarose gel. The DNA band was excised and the DNA fragmentpurified using a kit purchased from Qiagen.

[0553] The purified GM-CSF DNA fragment was digested with EcoRI andNgoM1. After digestion, the reaction mix was extracted withphenol:chloroform (1:1) followed by chloroform. The aqueous phase wasadjusted to 0.3M sodium acetate pH 5.2 and the DNA was precipitated with2 volumes of ethanol at −80° for 2 hours. The DNA was pelleted bycentrifugation, ethanol was removed, and the pellet was rinsed with 70%ethanol. The pellet was dried under vacuum.

[0554] The GM-CSF DNA was resuspended in sterile water and ligated topUC19-GPI 21 that had been digested with EcoRI-NgoM1. Ligation was forone hour at room temperature. PUC19 GPI 21 ligated to GM-CSF DNA wasused to transform competent AG-1 cells. Transformed AG-1 cells wereselected on LB plates with ampicillin. Plasmid DNA was isolated andanalyzed as above. Restriction digests were performed to confirm thepUC19 GPI-GM-CSF chimeric construct. The DNA from several positiveclones was isolated and sequenced.

[0555] This plasmid was digested with NgoMIV and KpnI, and the largerresulting fragment isolated after electrophoresis through a 1% agarosegel.

[0556] The 280 bp GPI modification signal sequence from the yeastprotein Gas1 was amplified by PCR from the yeast cosmid clone C9952(ATCC). This PCR employed pfu polymerase and the primers: UpstreamPrimer 5′GTAGCCGGCGCTAGCTCGGGGTCTTCTTCCAAGTCTA Downstream Primer5′TACGGTACCCCTAGGCCACAATGAAATAAGATACCATACC3′

[0557] These primers add a 5′ NgoMIV site and a 3′ KpnI site to the Gas1fragment. Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute Cycles 25 

[0558] The PCR product was purified after electrophoresis through a 1%agarose gel and digested with NgoM IV and KpnI. The Gas 1 GPI signalsequence was then ligated into the pUC19-GM-CSF-GPI plasmid preparedabove so that the Gas1 signal sequence was fused in-frame downstream ofthe GM-CSF sequence, replacing the Thy-1 sequence. This vector is termedpUC19-GMCSF-Gas1.1. The resultant plasmid was then transformed into AG-1competent E. coli (Stratagene) and plasmid clones were isolated byalkaline lysis mini-prep. Plamids were then screened for inserts byrestriction digest. DNA from a positive clone was sequenced to confirmthe identity of the GAS1 coding region.

[0559] A yeast expression plasmid for GPI-GM-CSF was then generatedutilizing the pITY-4 vector, which was kindly provided by Dr. K. DaneWittrup (University of Illinois). This plasmid stably integrates intothe yeast genome and allows high-level expression of heterologous genes.Features of pITY-4 include: a delta sequence (LTR of Ty element) thatenables multiple integration events by homologous recombination; aneo/kanamycin resistance gene that provides for selection in E.coli andtunable selection in yeast; the Gal1 promoter for high-level inducibletranscription; a unique EagI cloning site; a synthetic Pre-Pro sequenceoptimized for efficient secretion of expressed genes; the alpha factortermination sequence; and an origin of replication for propagation inE.coli. In this system, yeast are grown in dextrose-containing media for3 days, then are switched to media containing galactose to inducetranscription of genes inserted downstream of the Gal1 promoter.

[0560] The GMCSF-Gas1 insert described above was amplified by PCR frompUC19-GMCSF-Gas1.1 using pfu polymerase and the primers: Upstream5′TACGGCCGGCACCCACCCGCTCACCC3′ Downstream5′TACGGCCGCCACAATGAAAATAAGATACCAT3′

[0561] These primers add EagI sites at both ends for cloning into thepITY-4 plasmid. Conditions for PCR were: Denaturation 90° one minuteAnnealing 60° one minute Extension 72° one minute Cycles 25 

[0562] The PCR product was purified after electrophoresis through a 1%agarose gel and digested with EagI. The EagI-flanked GMCSF-Gas1 fragmentwas ligated into EagI-digested pITY-4 and used to transform E.coli AGIcells. E. coli were then grown on kanamycin-containing LB plates (100ug/ml). Plasmids from kanamycin resistant colonies were purified bymini-prep and mapped by restriction digests for presence and correctorientation of inserts. The identity of a positive clone was confirmedby sequencing. This plasmid is termed pITY-GMCSF-Gas1.1.

Example 2

[0563] Expression of Murine GM-CSF Fused to the Gas1 GPI ModificationSignal Sequence in Yeast

[0564] A 50 ml culture of the E.coli clone containing pITY-GMCSF-Gas1.1was grown in LB with 100 ug/ml kanamycin and the plasmid purified usinga Midi-Prep Kit from Qiagen. The S. cerevesiae strain BJ5464 (ATCC) wasthen transformed with pITY-GMCSF-Gas1.1 using a lithium acetate (LiAc)protocol. A 10 ml overnight culture of BJ5464 in YPD (Per liter: 20 gBactotryptone, 10 g yeast extract, 20 g dextrose) was used to inoculatea 100 ml flask. Yeast were grown for 3 hours at 300 and then harvestedby centrifugation at 12,000×g for 2 minutes at room temperature. Cellswere washed with sterile water and centrifuged again. The cells wereresuspended in 1.0 ml of 100 mM LiAc, transferred to a 1.5 ml microfugetube and centrifuged in an Eppendorf microfuge at top speed for 15seconds. The cells were then resuspended in 0.5 ml of 100 mM LiAc and 50uL samples were aliquoted to individual tubes. The cells were pelleted.240 uL of PEG (50% w/v), 36 uL 1.0M LiAc, 5 uL (10 mg/ml) boiled carrierDNA (salmon sperm DNA, Sigma), and 2 ug plasmid in 75 uL water, werethen added in that order. After the addition of plasmid, the tube wasvortexed, incubated at 300 for 30 minutes and heat-shocked at 42° for 15minutes. The cells were then pelleted, resuspended in sterile water andplated on YPD plates containing 1 mg/ml G418.

[0565] Individual colonies of G418-resistant yeast were picked and grownin one ml of YPD with 1 mg/ml G418 for 3 days. The cells were thenpelleted by centrifugation in a microfuge and the YPD(dextrose-containing, galactose-free) media was replaced with YPG (20 gbactotryptone, 10 g yeast extract, 20 g galactose per liter) with 1mg/ml G418. Yeast were grown in YPG for 3 days to allow full inductionof transcription from the Gal I promoter. After induction, cells werepelleted, washed with TN (0.15M NaCl, 25 mM Tris pH 7.4) and lysed in TNcontaining 20 mM octyl glucopyranoside (OGP), 1 mM PMSF, and 1 ug/mleach aprotinin, leupeptin and pepstatin. Yeast were lysed by vortexingwith acid-washed glass beads (425-600 microns, Sigma). Insolublematerial was pelleted and the supernatant assayed using a murine GM-CSFELISA (Endogen). A colony expressing high levels of GPI-GM-CSF wasidentified. Based on standard curve of soluble GMCSF, we estimateexpression to be approximately 25 ug/L, a significant improvement overmammalian expression and sufficient for in vivo experiments. This yeastclone is designated SC-GM-GPI.

[0566] One of the advantages of stably integrating vectors forexpression in yeast is that, after the initial cloning and colonyisolation, antibiotic maintenance is no longer required. To confirmthis, cells were grown with and without G418 and tested for GPI-GM-CSFexpression. We have seen no decrease in expression levels in the absenceof G418 over 8 months.

[0567] To produce GPI-GM-CSF on a scale suitable for in vitro and invivo functional characterization, 500 ml of YPD was inoculated withSC-GM-GPI and grown for three days at 30° with shaking. Cells werepelleted by centrifugation at 12,000×g for 2 minutes at room temperatureand transferred to an equal volume of YPG for an additional three daysof growth. Cells were then pelleted, washed with TN and lysed in 25 mlof TN containing 20 mM OGP, 1 mM PMSF, and 1 ug/ml each aprotinin,leupeptin and pepstatin. Cells were then lysed by vortexing with acidwashed glass beads, 20 g/500 ml culture, (425-600 microns, Sigma).Insoluble material was pelleted at 8,000×g for 10 minutes at roomtemperature and the soluble material was applied to an immunoaffinitycolumn of anti-murine GMCSF monoclonal antibody (Endogen) linked tocyanogen bromide-activated Sepharose 4B (Sigma). Coupling of themonoclonal to the Sepharose was performed according to themanufacturer's instructions. Efficiency of coupling was monitored usingOD₂₈₀ and binding of murine GM-CSF to immobolized antibody was confirmedusing commercially available, recombinant cytokine.

[0568] Soluble yeast-derived material was applied to the column andallowed to flow by gravity. The column was washed sequentially with: (a)20 volumes of TN with 1% Triton X-100; (b) 5 volumes of 50 mM Tris pH8.0, 1 mM OGP; (c) 20 volumes TN with 1 mM OGP. Bound material was theneluted with 10 volumes of 0.15M NaCl, 25 mM Tris pH 2.5 with 1 mM OGP.Eluted material was neutralized with 1/200 volume of 1.5M Tris pH8.8.The purified material was concentrated using a Microsep 3K centrifugaldevice (Pall Gelman Laboratory). Yields of GPI-GM-CSF were determined byELISA (Endogen) to be 25 ug/L of culture. Final concentration wasadjusted to 40 ug/ml by addition of 0.15M NaCl, 25 mM Tris pH 7.4 with 1mM OGP.

[0569] Purified GPI-GM-CSF was analyzed by stained gel and western blot.Approximately 1 ug of purified GPI-GM-CSF or recombinant soluble murineGM-CSF per lane were electrophoresed. Gels were then stained with silvernitrate using the Sigma silver staining kit according to themanufacturer's directions (Sigma). For western blots, gels weretransferred to Protran BA83 (Schleicher and Schuell) using an OwlScientific electric transblotter and blocked with TBS (Tris BufferedSaline) containing 0.05% Tween 20 and 2% nonfat dry milk overnight atroom temperature. The blot was then incubated with primary antibody (ratmonoclonal anti-murine GMCSF, Endogen) at 1:5000 dilution in blockingbuffer for 2 hours at room temperature. The blot was washed withTBS-0.05% Tween 20, and incubated with a secondary antibody, alkalinephosphatase conjugated goat anti-rat IgG (Sigma) at 1:10,000 for 1 hourat room temperature. After washing, color was developed with NBT-BCIP(Sigma). A single dominant band migrating at approximately the same rateas a recombinant soluble GM-CSF standard was clearly present on both thegel and the blot (the molecular weight of the GPI moiety is onlyapproximately 1500 compared to approximately 14,000 for the proteinmoiety]. Given the immunoreactivity with anti-GM-CSF and the ability ofthis material to bind to tumor cell membranes, these bands appear torepresent GPI-GM-CSF. While some high molecular weight material,possibly representing aggregates, is visible in the blot, this materialis not visible in the less sensitive silver stain, indicating that it ispresent in lower amount than the dominant band.

Example 3

[0570] Attachment of Murine GM-CSF Fused to the Gas1 GPI ModificationSignal Sequence to Cells

[0571] Wild type CMS-5 murine fibrosarcoma cells grown in DMEM, 10% FBS,Pen-Strep were harvested, washed twice with RPMI 1640 (LifeTechnologies) and resuspended in RPMI 1640 at a concentration of 5×10⁵cells/ml. 0.9 ml aliquots of the cell suspension were dispensed toEppendorf siliconized microfuge tubes. Each aliquot received either 1 ugof purified GPI-GM-CSF prepared as in Example 2, 1 ug of solublerecombinant murine GM-CSF (Intergen, supplied as lyophilized powder andreconstituted at 40 ug/ml in the same buffer as GPI-GM-CSF), or mediaalone. Cells were then incubated for 3 hours at 37° C. with shaking andthen washed 3 times with PBS containing 2% FBS.

[0572] For detection of GPI-GM-CSF by flow cytometry, cells wereincubated with a rat anti-murine GM-CSF monoclonal antibody (Endogen)for one hour at 40 C. The cells were then washed 3 times with PBScontaining 2% FBS, and incubated with FITC-labeled goat anti-rat IgGantibody (Sigma) for one hour at 4° C., and again washed 3 times withPBS containing 2% FBS. The cells were analyzed by flow cytometry on aBecton-Dickinson Facscalibur. Decoration with GPI-GM-CSF caused anapproximately 10-fold increase in peak and mean FL-1 fluorescencerelative to cells incubated with media alone. In contrast, cellsincubated with soluble GM-CSF had virtually the same profile as thenegative control cells. This data indicates that GPI-GM-CSF, but notsoluble recombinant GM-CSF, can bind to tumor cells.

[0573] GPI-GM-CSF attached to CMS-5 cells was also detected andquantitated by ELISA. CMS-5 cells were harvested and washed as describedabove. 1×10⁶ cells in 1 ml of RPMI 1640 were incubated with 1 ug ofpurified GPI-GM-CSF. After incubation for 2 hours at 37° C., the cellswere washed 3 times with PBS containing 2% FBS. The cell pellet waslysed with 50 microliters of PBS containing 0.15% deoxycholate and thedetergent subsequently diluted by the addition of 200 microliters ofPBS. The material was serially diluted with PBS and amounts of GM-CSFdetermined using an ELISA kit (Endogen) against a soluble, recombinantGM-CSF standard provided by the manufacturer. Based on this data, themean number of GPI-GM-CSF molecules incorporated/cell over fiveexperiments was 37,000+/−33,000. The large standard deviation was due toone experiment in which the number of molecules/cell was 66,000.Excluding this experiment, the mean was 29,500+/−4,500.

[0574] “Decoration” of B16 murine melanoma cells with GPI-GM-CSF wasalso quantitated by ELISA. Decoration and ELISA were performed exactlyas described for CMS-5 cells. The mean number of molecules/cell overthree experiments was 21,000+/−11,500.

Example 4

[0575] Stability of Murine GM-CSF Fused to the Gas1 GPI ModificationSignal Sequence on Cells

[0576] To study the stability of incorporated GPI-GM-CSF on cells, CMS-5cells were harvested and decorated as described above in Example 3.After decoration, the cells were washed 3 times with PBS containing 2%FBS and then resuspended at 4×10⁶ cells/ml in RPMI 1640. The cells wereirradiated at 3500 rads from a ¹³⁷Cs source. The cells were thenincubated at 37° C. in 5% CO₂ and aliquots were removed at hourlyintervals, washed three times, and lysed in 50 ul PBS with 0.15%deoxycholate. 200 ul of PBS was then added to dilute the deoxycholate.Cell-associated GM-CSF was measured by ELISA. Even at 6 hours, cellsshowed only about a 20% loss of cell-surface GPI-GM-CSF. After 6 hours,viability of irradiated cells (both decorated and non-decorated) asmeasured by microscopic inspection with trypan blue staining wassignificantly compromised. However, in vivo data (see below) indicatesthat both cell-surface retention of GPI-GM-CSF and post-irradiationcellular viability are sufficient to sustain a biological effect.

Example 5

[0577] Bioactivity of Murine GM-CSF Fused to the Gas1 GPI ModificationSignal Sequence to Cells

[0578] The bioactivity of GPI-GM-CSF was assayed by determining themolecule's ability to support the proliferation of the FDC-P 1 cellline, a murine bone-marrow derived, GM-CSF dependent cell line.Proliferation of FDC cells was measured with the Biotrak CellProliferation ELISA (Amersham Pharmacia), an assay that utilizes thethymidine analogue 5-bromo-2′-deoxyuridine (BrdU). WEHI cells (ATCC)were grown in Iscove MEM, 10% FBS, Penicillin-streptomycin, as a sourceof conditioned media for FDC-P1 cells. FDC-P1 cells were grown in DMEM,10% FBS, Penicillin-streptomycin with 25% WEHI conditioned media,harvested, and washed 3 times with DMEM, 10% FBS,Penicillin-streptomycin. The cells were resuspended at 1×10⁵/ml in DMEM,10% FBS, Penicillin-streptomycin and 100 uL was aliquoted to individualwells of a 96 well microtitre plate. Groups, done in triplicate, were asfollows:

[0579] A. Media-No Cells

[0580] B. FDC-PI Cells-Unstimulated

[0581] C. FDC-P1 Cells+10 ng soluble GM-CSF

[0582] D. FDC-PI Cells+10 ng GM-CSF as GPI-GM-CSF (as determined byELISA against GM-CSF standard)

[0583] E. FDC-PI Cells+10 ng GPI-GM-CSF (as in “D”) denatured byextraction of the protein with chloroform:methanol (3:1) followed byacetone precipitation and resuspension.

[0584] All protein solutions were diluted to 100 ng/ml in 0.15M NaCl, 25mM Tris pH 7.4 with 1 mM OGP, so that the volume added to each well was100 uL.

[0585] The non-isotopic proliferation assay was performed according tothe manufacturer's instructions. The plated cells were grown for twodays at 37° C. in 5% CO₂. On day 3, 10 ul of the BrdU solution was addedto individual wells and the cells incubated for 3 more hours. The platewas then centrifuged at 300×g for 10 minutes and the supernatantremoved. The plate was dried by incubating at 60° C. for one hour. Theplate was then fixed and blocked according to the manufacturer'sinstructions. The fixed cells were then incubated withperoxidase-labelled anti-BrdU for 90 minutes. The wells were then washedand color developed with TMB according to the manufacturer'sinstructions. In two experiments, GPI-GM-CSF consistently sustainedproliferation at a level somewhat (about 25%) higher than did solublerecombinant GM-CSF, indicating that GPI-GM-CSF is suprabioactive. Theeffect of GPI-GM-CSF was not due to the GPI moiety alone, sincedenatured GPI-GM-CSF did not support proliferation. The GPI moietyremained linked to the protein after denaturation, since the protein wasstill able to decorate cells as demonstrated by ELISA, which recognizeslinear epitopes on GM-CSF.

Example 6

[0586] Effective Immunization with Cells Admixed with GPI-GM-CSF

[0587] These experiments included mice vaccinated with:

[0588] (a) Wild-type cells (WT)

[0589] (b) Cells incubated with soluble GM-CSF-Unwashed (total GM-CSF indose: 1 microgram)

[0590] (c) Cells decorated with GPI-GM-CSF-Unbound GPI-GM-CSF Washed offFollowing Incubation (total GPI-GM-CSF in dose: 0.74 nanograms by ELISA[mean of 2 experiments; 73 and 75 ng individually)

[0591] (d) Cells decorated with GPI-GM-CSF-Unwashed (total GM-CSF indose: 1 microgram)

[0592] GPI-GM-CSF mass and concentration values are expressed in termsof equivalence to GM-CSF as determined by ELISA against a soluble GM-CSFstandard.

[0593] CMS-5 cells were grown to 70% confluence in DMEM, 10% FBS,Penicillin-streptomycin, harvested trypsinization, and washed 3 timeswith RPMI 1640. Viability was determined by trypan blue staining of analiquot and the cells were then resuspended at a concentration of 4×10⁶cells/ml a 1 ul aliquots dispensed into siliconized microfuge tubes. Thecells were incubated with 1 ug GPI-GM-CSF or 1 ug soluble recombinantmurine GM-CSF per 10⁶ cells for 3 hours at 37° C. “Washed” groups werethen washed 3 times with PBS, 2% FBS and resuspended at 4×10⁶ cells/mlin RPMI 1640. An aliquot of the washed GPI-GM-CSF decorated cells wasremoved and the amount of cell-associated GM-CSF measured by ELISA asdescribed above. There were approximately 31,000 and 32,000 GPI-GM-CSFmolecules/cell in the washed decorated groups in the two experiments,respectively.

[0594] The cells were irradiated at 3500 rads from a ¹³⁷Cs source. 8-10week-old female Balb/c mice (which are syngeneic for CMS-5) wereanesthetized by metofane inhalation and vaccinated subcutaneously in theleft inguinal fold with 1×10⁶ cells in 0.25 ml. Seven days later,wild-type CMS-5 cells at 70% confluence were harvested and washed 3times in HBSS. Viability was determined by trypan blue staining of analiquot and cells were adjusted to 4×10⁶/ml in HBSS. The previouslyvaccinated mice were then injected subcutaneously behind the neck, undermetofane anesthesia, with 2×10⁶ live, wild-type CMS-5 cells in 0.5 mlHBSS.

[0595] Tumor development was assessed daily by palpation and visualinspection. “Onset” was defined as the first day on which a tumor masswas both palpable and visible. The observer was blinded to the vaccinereceived by each set of mice to ensure against bias. Mice weresacrificed by CO2 asphyxiation when tumors become unwieldy. Experimentswere terminated 70 days after tumor challenge, as planned in advance.

[0596] Data is pooled from three experiments for GPI-GM-CSF unwashed,soluble GM-CSF, and wild-type vaccine groups. Data for these groupsincludes that from undepleted controls in a lymphocyte subset depletionexperiment. Data for the GPI-GM-CSF washed group is pooled from twoexperiments, since this group was not included in the initial depletionexperiment. The depletion experiment had 4 mice/group, and the otherexperiments had 5/group. In terms of total mouse numbers, n=14 forGPI-GM-CSF unwashed; 10 for GPI-GM-CSF washed; 14 for soluble GM-CSFunwashed; and 14 for WT. Approximate percentages of mice survivingtumor-free to day 70 after challenge were: WT, 15%; soluble GM-CSF, 50%;GPI-GM-CSF washed, 60%; GPI-GM-CSF unwashed, 85%. Thus, even though theGPI-GM-CSF washed vaccine contained over a thousand-fold less GM-CSFthan the unwashed soluble, administration of cells decorated withGPI-GM-CSF was more effective. Furthermore, the GPI-GM-CSF unwashedvaccine, in which some molecules were not attached to a cell, was evenmore effective.

Example 7

[0597] Cloning and Expression of Human GM-CSF Fused to the Gas1 GPIModification Signal Sequence

[0598] Human GM-CSF is amplified by PCR from a human T cell cDNA library(Clontech) using Pfu polymerase (Stratagene). The following primers areused: Upstream 5′GCGAATCCCGGCCGGCACCCGCCCGCTCGCCCAGCCCC Downstream5′CAGCCGGCCTCCTGGACTGGCTCCCAGCAGTC

[0599] The upstream primer contains EcoR1 and Eag1 restriction sitesimmediately preceding the first amino acid found in the mature humanGM-CSF protein. Since expression in S. cerevisiae utilizes a yeastleader sequence, cloning of the human GM-CSF begins at the N terminus ofthe mature protein. Each downstream primer omits the native stop codonto allow in-frame ligation to the sequence encoding the Gas1 GPImodification signal. The downstream primer contains an NgoM IVrestriction site, consistent with restriction sites used in otherconstructs.

[0600] PCR parameters are denaturation at 97° C. for 1 minute, annealingat 56° C. for 1 minute, and extension at 72° C. for 2 minutes.

[0601] PCR is performed for the least number of cycles yielding avisible band on agarose gel electrophoresis. After amplification, thereaction mix is allowed to cool at 4° for 10 minutes.

[0602] The PCR product is isolated by electrophoresis through a 1%agarose gel and eluted from the excised agarose band using acommercially available kit (Qiagen). The purified hGM-CSF DNA fragmentis digested with EcoR1 and NgoM IV and ligated to the (murine) pUC19GMCSF-GPI plasmid that has been digested with EcoR1 and NgoM IV. Thisreplaces the murine GM-CSF with its human counterpart. The pUC19-hGM-CSFGPI plasmid is then transformed into competent AG-1 E. coli cells, 30colonies are picked for mini-culture, and plasmid clones are isolatedand purified using commercially available kits (Qiagen). Positive clonesare identified by restriction enzyme test digest and agarose gelelectrophoresis. Positive E. coli colonies are grown overnight inmaxi-culture and their plasmids purified using Qiagen maxi-prep kits.Inserts are sequenced.

[0603] To clone GM-CSF GAS1 g into the pITY-4 expression vector, PCR ofthis construct from the pUC19 vector is performed. The primers are:5′TACGGCCGGCACCCGCCCGCTCGCCCAGCCCC 3′TACGGCCGCCACAATGAAAATAAGATACCAT

[0604] The upstream primer has an EagI site immediately preceding thefirst codon of the mature GM-CSF. This removes the mammalian secretionsignal and allows for in-frame ligation to the yeast signal sequence.The same restriction site can be used as for the mouse construct becauseit is absent in the human sequence. The downstream primer appends anEagI site at the 3′ end. PCR is performed using Pfu polymerase for 25cycles. Conditions for PCR are: denaturation 90° one minute, annealing60° one minute, extension 72° one minute. After amplification, thereaction mix is allowed to cool at 4° for 10 minutes.

[0605] The PCR product is isolated by electrophoresis through a 1%agarose gel and eluted from the excised agarose band using acommercially available kit (Qiagen). The purified GM-CSF GAS1 g DNAfragment is digested with EagI, ligated to pITY-4, and transformed intoAG-1 chemically competent bacteria (Stratagene). 30 colonies are pickedfor mini-culture and plasmid clones are isolated and purified usingcommercially available kits (Qiagen). Positive clones are identified byrestriction enzyme test digest and agarose gel electrophoresis. PositiveE. coli colonies are grown overnight in maxi-culture and their plasmidspurified using Qiagen maxi-prep kits. Inserts are sequenced.

[0606] The GPI-human GM-CSF molecule is expressed in S. cerevesiae asdescribed for the murine molecule in Example 2. Immunoaffinitypurification is performed as described in Example 2, substituting ananti-human GM-CSF antibody for the anti-murine GM-CSF antibody. ELISA todetect and quantitate the molecule, whether in isolation or bound to anantigen bearing target, is performed using an anti-human GM-CSFmonoclonal antibody, as is flow cytometry on cells decorated with themolecule.

Example 8

[0607] Cloning of GM-CSF/Influenza Hemagglutinin Chimeric Proteins

[0608] pUC19 GMCSF-K-GAS1.1

[0609] pUC19 GM-CSF-K-HA was cloned starting with pUC19 GM-CSF-K-Gas1.1,which we produced in our laboratory. This plasmid includes a sequencethat encodes murine GM-CSF fused to a downstreamglycosylphosphatidylinositol modification sequence derived from theyeast GAS1 protein (the latter obtained from Dr. D. Wittrup, Universityof Illinois). A linker sequence is interposed between the GM-CSF andGAS1 portions. To insert the linker sequence, the plasmid pUC19GMCSF-Gas1.1, also previously produced in our lab, was digested withNgoM IV and NheI. These restriction enzymes cut at the 3′ end of theGM-CSF molecule and at the 5′ end of the Gas 1.1 sequence, respectively.The resulting plasmid was purified after electrophoresis through agarosegel using a kit manufactured by Qiagen. The following oligonucleotideswere purchased: 5′CCGGCACTAGTGGCGGAGGGGGCTCCGGCGGCGGGGGCAGCG5′CTAGCGCTGCCCCCGCCGCCGGCGCCCCCTCCGCCACTAGTG

[0610] The synthetic oligonucleotides contain:

[0611] 1. 5′ overhang that anneals to NgoM IV digested plasmid DNA

[0612] 2. 3′ overhang that anneals to Nhe I digested plasmid DNA

[0613] 3. DNA sequence coding for the peptide GGGGSGGGGS where G standsfor glycine and S stands for serine. This 10 amino acid sequence (G₄S)₂is designed to insert a kink/spacer in the protein between the GMCSF andthe Gas1.1 moieties.

[0614] 4. SpeI site to allow confirmation of cloning of the smallfragment and for further manipulations.

[0615] The two oligonucleotides were mixed in equimolar concentrations,boiled for 2 minutes and allowed to anneal at room temperature. Theoligonucelotide was ligated into the NgoM IV-Nhe I digested plasmid andthe plasmid was used to transform the E.coli strain AG-1. Transformantswere selected on LB plates containing 100 ug/ml ampicillin. Plasmid DNAwas isolated, digested with Spe I, and electrophoresed on agarose gelsto confirm the presence of the (G₄S)₂ sequence.

[0616] pUC19 GM-CSF-K-hemagglutinin (HA)

[0617] The plasmid pUC19 GM-CSF-K-HA was produced, which encodes achimeric protein containing (from amino terminal to carboxy terminal):(1) murine GM-CSF; (2) the (G₄S)₂ linker described above; and (3) theHA1 domain of the H1 HA from the A/PR/8/34 influenza A isolate. The HA1sequence used (amino acids 18 to 344 of the HA precursor) omits theN-terminal leader sequence and the downstream HA2 domain. A terminationcodon was added after amino acid 344.

[0618] pUC19 GM-CSF-K-Gas1.1. was digested with Nhe I and Kpn I. Nhe Icuts at the 5′ end of the Gas1.1 coding sequence and Kpn I cuts at the3′ end of the Gas1.1 coding sequence, respectively. The resultingplasmid with the GPI coding region removed, was purified afterelectrophoresis through agarose gel using a kit manufactured by Qiagen.The HA1 coding sequence was cloned by PCR from a plasmid encoding the HAgene of the A/PR/8/34 strain of influenza. The HA1 sequence used beginsat amino acid 18, the start of the mature protein, i.e. lacking thesecretion signal sequence. The 3′ end corresponds to amino acid 344,eliminating the transmembrane region and substituting a terminationcodon. Primers for PCR of the HA1 sequence were as follows: Upstream HA1Primer 5′ ATGCTAGCGACACAATATGTATAGGC Downstream HA1 Primer5′ ATGGTACCCGGCCGTTATCATCTGGATTGAATGGACGG

[0619] Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute

[0620] PCR was performed for 20 cycles using vent polymerase.

[0621] Following PCR, the product was electrophoresed through a 1.0%agarose gel and the HA1 cDNA was extracted from the gel using a Qiagenkit according to the manufacturer's instructions. The purified HA1 DNAfragment was digested with Nhe I and Kpn I. To make the fusion protein,the purified Nhe I-Kpn I HA fragment was ligated into the pUC19GM-CSF-K-Gas1.1 vector that had been digested with Nhe I and Kpn I toremove the Gas1.1 coding region. The DNA was used to transform E.coliAG1 and transformants selected on LB-ampicillin plates. Plasmid DNA fromindividual colonies was isolated and digested with restriction enzymes.Restriction digests identified a pUC19 GM-CSF-K-HA plasmid.

[0622] The pUC19 GM-CSF-K-HA plasmid was purified according to themanufacturer's instructions using a kit purchased from Qiagen.

Example 9

[0623] Cloning of GM-CSF-K-HA into Yeast Expression Vector

[0624] PCR of pUC19 GM-CSF-K-HA was used to isolate a DNA fragmentencoding GM-CSFK-HA for cloning into a yeast expression vector. The PCRproduct contains Eag I cloning sites for in frame insertion into theyeast expression vector. Upstream Primer 5′ TACGGCCGGCACCCACCCGCTCACCCDownstream Primer 5′ ATGGTACCCGGCCGTTATCATCTGGATTGAATGGACGG

[0625] Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute

[0626] PCR was performed for 20 cycles using vent polymerase.

[0627] Following PCR, the product was electrophoresed through a 1.0%agarose gel and the GM-CSF-K-HA gene was extracted from the gel using aQiagen kit according to the manufacturers instructions. The purified DNAfragment was digested with Eag I and ligated to the yeast expressionvector ITK that had been digested with Eag I. The ITK vector is designedfor (1) replication in E.coli and (2) expression of genes in the yeastSaccharomyces cerevisiae after stable integration using homologousrecombination. The vector contains:

[0628] 1. Sequences for replication of the plasmid in E.coli

[0629] 2. Yeast Gal promoter for expression of heterologous genes inyeast grown in media containing galactose.

[0630] 3. PrePro—Synthetic DNA sequence, optimized for secretion andsignal sequence cleavage of distal genes in yeast.

[0631] 4. Unique Eag I site for cloning genes to be expressed.

[0632] 5. Alpha terminator—DNA sequence for efficient termination ofproximal genes.

[0633] 6. Delta sequence that allows for stable integration of theplasmid by recombination with endogenous delta sequences in the yeastchromosome.

[0634] 7. Antibiotic resistance gene allowing for selection in E.coliwith kanamycin and selection in yeast with G418.

[0635] This plasmid was used to transform E.coli strain AG-1.Transformants were selected by growth on LB plates containing 100 ug/mlkanamycin. Individual colonies were grown in LB media containingkanamycin and plasmids were purified. Restriction digests determinedorientation of inserts. The resulting plasmid ITK GM-CSF-K-HA waspurified using a kit purchased from Qiagen according to themanufacturer's instructions.

Example 10

[0636] Expression of GM-CSF-K-HA in Yeast

[0637] The purified plasmid was linearized with Mfe 1 and used totransform the yeast strain Saccharomyces cerevisiae WDHY131 usinglithium acetate (LiAc). A 10 ml culture of S.cerevisiae grown tosaturation at 30° in YPD media (per liter/20 g Bactotryptone; 20 gdextrose; 10 g yeast extract) was used to inoculate 100 ml of YPD. Theculture was grown at 30° with shaking for 3 hours. The yeast wereharvested by centrifugation at 11,000×g for 2 minutes and resuspended in25 ml of sterile water. The yeast were centrifuged as above andresuspended in 1.0 ml of 100 mM lithium acetate and transferred to a 1.5ml microfuge tube. The yeast were pelleted by centrifugation at 12,000×gfor 15 seconds and the supernatant removed. The cells were resuspendedin 0.5 ml of 100 nM LiAc. 50 uL of cell suspension was added toindividual microfuge tubes and centrifuged as above. Supernatant wasremoved.

[0638] Transformation mix added to the yeast pellet consisted of: 240 uLPEG (50% w/v); 36 uL 1.0 M LiAc; 5 uL single stranded DNA (10 mg/ml) and1 ug of linearized ITK GM-CSF-K-HA in 75 uL of water. The mixture wasvortexed to resuspend the cell pellet and incubated at 30° for 30minutes. The cells were then shocked at 42° for 15 minutes, centrifugedto pellet cells and resuspended in 0.5 ml of YPD. Yeast were incubatedin YPD media for 3 hours and plated on YPD plates containing 2 mg/mlG418. Plates were grown at 30° for 3 days until individual coloniesappeared. To screen for expression of GM-CSF-K-HA, individual colonieswere grown in 1 ml of YPD media at 30° for 2 days. The cells werecentrifuged at 8,000×g for 2 minutes and the YPD media removed andreplaced with 1 ml of YPG media (per liter/20 g Bactotryptone; 20 ggalactose; 10 g yeast extract) for induction from the gal promoter.Yeast were grown in YPG media for 2 days. At this time, an aliquot wasremoved and cells were pelleted. The supernatant was tested for GM-CSFexpression using an ELISA kit purchased from Endogen. Protocol wasaccording to the manufacturer. A high-expressing yeast clone secretingthe chimeric protein GM-CSF-K-HA was identified. Based on standard curveof soluble GMCSF, expression level was approximately 2.4 mg/L of GM-CSFmoiety.

Example 11

[0639] Production of pUC19 HA (hemagglutinin)-K-GM-CSF

[0640] The plasmid pUC19 HA-K-GM-CSF was also produced, which encodes achimeric protein containing (from amino terminal to carboxy terminal):(1) an HA1 domain (2) K, the (G₄S)₂ linker described above, and (3)murine GM-CSF, The HA1 begins at the amino terminus of the matureprotein, amino acid 18, eliminating the leader sequence. The 3′ endterminates at amino acid 344. The (G₄S)₂ has been added to supply aflexible linker. The GM-CSF begins at amino acid 18 of the GM-CSFprotein, corresponding to the first amino acid of the mature protein.

[0641] The HA-K sequence was first cloned by PCR of the HA1 codingsequence from a plasmid encoding the HA gene of the A/PR/8/34 strain ofinfluenza. Upstream Primer 5′CTGAATTCCGGCCGGACACAATATGTATAGGC DownstreamPrimer 5′ATGGTACCGCTGCCCCCGCCGCCGGAGCCCCCTCCGCCACTTCTGGATTGAATGGACGGAAT

[0642] The oligonucleotides for PCR generate a nucleic acid with:

[0643] 1. A 5′ EcoRI site at the amino terminus of the mature HA

[0644] 2. The (G₄S)₂ linker at the carboxy terminus of the HA1 domain(amino acid 344 of the HA precursor)

[0645] 3. A Kpn I site distal to the end of the (G₄S)₂ sequence

[0646] Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute

[0647] PCR was performed for 20 cycles using vent polymerase.

[0648] Following PCR, the product was electrophoresed through a 1.0%agarose gel and the HA1-K DNA was extracted from the gel using a Qiagenkit according to the manufacturer's instructions. The purified HA-K DNAfragment was digested with EcoRI and Kpn I and the fragment was clonedinto pUC19 that had been digested with EcoRI and KpnI. The plasmid wasused to transform E.coli AG-1 to amp. Individual colonies were pickedand grown in LB-amp. The identity of plasmids with the correct insertwas determined by restriction mapping. The resulting plasmid termedpUC19 HA-K was purified using a Qiagen kit according to themanufacturer's instructions.

[0649] The GM-CSF fragment was cloned by PCR. Upstream Primer5′ ACGGTACCGCACCCACCCGCTCACCCATC Downstream Primer5′ TAGGATCCCGGCCGTCATTTTTGGACTGGTTTTTTGCACG

[0650] The PCR primers generate a GM-CSF fragment with

[0651] 1. 5′ KpnI site that allows in frame translation from the (G₄S)₂portion of the HA-K molecule to the start of the mature GM-CSF moleculeat amino acid 18.

[0652] 2. A termination codon at the 3′ end of the GM-CSF

[0653] 3. 3′ BamHI site

[0654] Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute

[0655] PCR was performed for 20 cycles using vent polymerase.

[0656] Following PCR, the product was electrophoresed through a 1.0%agarose gel and the GM-CSF gene was extracted from the gel using aQiagen kit according to the manufacturer's instructions. The purifiedfragment was digested with Kpn I and BamHI and the fragment was ligatedinto pUC19 HA-K plasmid that had been digested with KpnI and BamHI. Theplasmid was used to transform E.coli AG-1 to amp. Individual colonieswere picked and grown in LB-amp. The identity of plasmids with thecorrect insert was determined by restriction mapping. The resultingplasmid termed pUC19 HA-K-GM-CSF was purified using a Qiagen kitaccording to the manufacturer's instructions.

Example 12

[0657] Cloning of HA-K-GM-CSF into Yeast Expression Vector

[0658] PCR of pUC19 HA-K-GM-CSF was used to generate a DNA fragmentencoding HA-K-GM-CSF for cloning into a yeast expression vector. The PCRproduct contains Eag I cloning sites for in-frame insertion into theyeast expression vector. Upstream Primer5′CTGAATTCCGGCCGGACACAATATGTATAGGC Downstream Primer5′TAGGATCCCGGCCGTCATTTTTGGACTGGTTTTTTGCACG

[0659] Conditions for PCR were: Denaturation 90° one minute Annealing60° one minute Extension 72° one minute

[0660] PCR was performed for 20 cycles using vent polymerase.

[0661] Following PCR, the product was electrophoresed through a 1.0%agarose gel and the HA-K-GM-CSF gene was extracted from the gel using aQiagen kit according to the manufacturers instructions. The purified DNAfragment was digested with Eag I and ligated to the yeast expressionvector ITK, that had been digested with Eag I. The ITK vector isdesigned for (1) replication in E.coli and (2) expression of genes inthe yeast Saccharomyces cerevisiae after stable integration usinghomologous recombination. The vector contains:

[0662] 1. Sequences for replication of the plasmid in E.coli

[0663] 2. Yeast Gal promoter for expression of heterologous genes inmedia containing galactose.

[0664] 3. PrePro—Synthetic DNA sequence, optimized for secretion andsignal sequence cleavage of distal genes in yeast.

[0665] 4. Unique Eag I site for cloning genes to be expressed.

[0666] 5. Alpha terminator—DNA sequence for efficient termination ofproximal genes.

[0667] 6. Delta sequence that allows for stable integration of theplasmid by recombination with endogenous delta sequences in the yeastchromosome.

[0668] 7. Antibiotic resistance gene allowing for selection in E.coliwith kanamycin and selection in yeast with G418.

[0669] This plasmid was used to transform E.coli strain AG-1.Transformants were selected by growth on LB plates containing 100 ug/mlkanamycin. Individual colonies were grown in LB media containingkanamycin and plasmids were purified. Restriction digests determinedorientation of inserts. The resulting plasmid ITK HA-K-GM-CSF waspurified using a kit purchased from Qiagen according to themanufacturer's instructions.

[0670] The purified plasmid was linearized with Mfe 1 and used totransform the yeast strain Saccharomyces cerevisiae WDHY131 usinglithium acetate (LiAc). A 10 ml culture of S.cerevisiae grown tosaturation at 30° C. in YPD media (per liter/20 g Bactotryptone; 20 gdextrose; 10 g yeast extract) was used to inoculate 100 ml of YPD. Theculture was grown at 30° C. with shaking for 3 hours. The yeast wereharvested by centrifugation at 11,000×g for 2 minutes and resuspended in25 ml of sterile water. The yeast were centrifuged as above andresuspended in 1.0 ml of 100 mM lithium acetate and transferred to a 1.5ml microfuge tube. The yeast were pelleted by centrifugation at 12,000×gfor 15 seconds and the supernatant removed. The cells were resuspendedin 0.5 ml of 100 mM LiAc. 50 uL of cell suspension was added toindividual microfuge tubes and centrifuged as above. Supernatant wasremoved. Transformation mix added to the yeast pellet consisted of: 240uL PEG (50% w/v); 36 uL 1.0 M LiAc; 5 uL single stranded DNA (10 mg/ml)and 1 ug of linearized ITK HA-K-GM-CSF in 75 uL of water. The mixturewas vortexed to resuspend the cell pellet and incubated at 30° for 30minutes. The cells were then shocked at 42° C. for 15 minutes,centrifuged to pellet cells and resuspended in 0.5 ml of YPD. Yeast wereincubated in YPD media for 3 hours and plated on YPD plates containing 2mg/ml G418. Plates were grown at 30° C. for 3 days until individualcolonies appeared. To screen for expression of HA-K-GM-CSF, individualcolonies were grown in 1 ml of YPD media at 30° C. for 2 days. The cellswere centrifuged at 8,000×g for 2 minutes and the YPD media removed andreplaced with 1 ml of YPG media (per liter/20 g Bactotryptone; 20 ggalactose; 10 g yeast extract) for induction from the gal promoter.Yeast were grown in YPG media for 2 days. At this time, an aliquot wasremoved and cells were pelleted. The supernatant was tested for GM-CSFexpression using an ELISA kit purchased from Endogen. The protocol wasaccording to the manufacturer.

[0671] A colony expressing high levels of the chimeric protein wasidentified. Based on standard curve of soluble GMCSF, expression levelis approximately 2.0 mg/L of soluble material. There is no decrease inexpression levels in the absence of G418.

Example 13

[0672] Scale Up Purification of GM-CSF-K-HA

[0673] For scaled-up purification of the chimeric protein, yeast wereinoculated into 500 ml of YPD and grown for three days at 30° C. Cellswere pelleted by centrifugation at 12,000×g for 2 minutes andtransferred to an equal volume of YPG for an additional three days ofgrowth. The cells were then pelleted by centrifugation at 12,000×g for 2minutes and the supernatant collected. The soluble material was appliedto an immunoaffinity column of anti-murine GMCSF monoclonal antibody(Endogen) linked to cyanogen bromide-activated Sepharose 4B (Sigma).Coupling of the monoclonal to the Sepharose was performed according tothe manufacturer. Efficiency of coupling was monitored using OD₂₈₀ andof binding of GMCSF to immobolized antibody was tested using soluble,commercially available material.

[0674] Soluble yeast-derived material was applied to the column andallowed to flow by gravity. The column was washed with: (a) 20 volumesof 0.15M NaCl, 25 mM Tris pH 7.4 (TN) (b) 5 volumes of 50 mM Tris pH 8.0(c) 20 volumes TN. Bound material was then eluted with 10 volumes of0.15M NaCl, 25 mM Tris pH 2.5. Eluted material was neutralized with1/200 volume of 1.5M Tris pH8.8. The purified material was concentratedusing a Microsep 3K centrifugal devise (Pall Gelman Laboratory). Yieldsof chimeric protein were determined by ELISA (Endogen) according to themanufacturer's instructions.

[0675] Purified GM-CSF-K-HA

[0676] Purified GM-CSF-K-HA was analyzed by western blot. Approximatelylug of GM-KHA per lane was electrophoresed along with soluble GMCSF. Forwestern blot, gels were transferred to Protran BA83 (Schleicher andSchuell), blocked with TBS (Tris Buffered Saline) containing 0.05% Tween20 and 2% Nonfat Dry Milk. The blot was incubated with primary antibody(rat monoclonal anti-murine GMCSF, Endogen) at 1:5000 dilution inblocking buffer for 2 hours at room temperature. The blot was washedwith TBS-0.05% Tween 20. Secondary antibody, alkaline phosphataseconjugated anti-rat IgG (Sigma) was incubated at 1:10,000 for 1 hour atroom temperature.

Example 14

[0677] Decoration of Cells with GM-K-HA

[0678] Purified GM-CSF-K-HA was used to decorate CMS 5 murinefibrosarcoma cells. CMS 5 cells were grown in DMEM, 10% FBS,Penicillin-streptomycin, harvested by trypsinization and washed 3 timeswith RPMI 1640 (Gibco). Cells were diluted to 1×10⁶/ml in RPMI 1640 and0.9 ml were aliquoted to siliconized tubes. Cells were incubated for 2hours at 37° C. with shaking and then washed 3 times with PBS containing2% FBS. Primary antibody, rat anti-murine GMCSF monoclonal, wasincubated for one hour at 4° C. Cells were washed as above, treated withFITC labeled anti-rat antibody (Sigma), and incubated for one hour at 4°C. After additional washing, the cells were analyzed by flow cytometry,which confirmed the presence of GM-CSFK-HA on the surface of the tumorcells.

Example 15

[0679] Quantitation of GM-CSF-K-HA on the Cell Surface of CMS 5 CellsAfter Decoration

[0680] CMS 5 cells were harvested and washed as described above. 1×10⁶cells in 1 ml of RPMI 1640 were incubated with 1 ug of purified GM-K-HA.After incubation for 15 min, 30 min, 1 hour, or 2 hours at 4° C., roomtemperature, or 37° C., the cells were washed 3 times with PBScontaining 2% FBS. The cell pellet was lysed with 50 microliters of PBScontaining 0.15% deoxycholate and the detergent subsequently diluted bythe addition of 200 microliters of PBS. The material was seriallydiluted with PBS and tested by ELISA (Endogen). Based on the amount ofGM-CSF detected in the cell lysates, it was possible to quantitate theaverage number of GM-CSF molecules associated with each cell. Forexample, after a 15 min incubation at 4° C., 58,700 molecules werepresent per cell. After a 15 min incubation at room temperature, 25,700molecules were present per cell. After a 15 min incubation at 37° C.,17,200 molecules were present per cell.

Example 16

[0681] Effective Immunization with Tumor Cells Admixed withGM-CSF/Hemagglutinin Fusion Polypeptides

[0682] CMS-5 murine fibrosarcoma cells were grown to 70% confluence inDMEM, 10% FBS, Penicillin-streptomycin, harvested by trypsinization, andwashed 3 times with RPMI 1640. Viability was determined by trypan bluestaining of an aliquot and the cells were then resuspended at aconcentration of 4×10⁶ cells/ml and 1 ml aliquots dispensed intosiliconized microfuge tubes. The cells were incubated with 1 ug(microgram) murine GM-CSF-K-HA or 10 ng (nanograms) HA-K-murine GM-CSFper 10⁶ cells for 3 hours at 37° C. Cells were then washed 3 times withRPMI 1640 and resuspended at 4×10⁶ cells/ml in RPMI 1640. An aliquot ofthe cells was removed and the amount of cell-associated GM-CSF measuredby ELISA as described above. There were approximately 20,240 and 18,000molecules/cell in the GM-CSF-K-HA and HA-K-GM-CSF groups, respectively.Cells for a control vaccine, to be administered without a molecule ofthe invention (or any other immunomodulator), were prepared in parallel.

[0683] The cells were irradiated at 3500 rads from a ¹³⁷Cs source. 8week-old female Balb/c mice (which are syngeneic for CMS-5) wereanesthetized by metofane inhalation and vaccinated subcutaneously in theleft inguinal fold with 1×10⁶ cells in 0.25 ml. Each mouse receivedcells from only one vaccine type. Seven days later, wild-type CMS-5cells at 70% confluence were harvested and washed 3 times in HBSS.Viability was determined by trypan blue staining of an aliquot and cellswere adjusted to 4×10⁶/ml in HBSS. The previously vaccinated mice werethen injected subcutaneously behind the neck, under metofane anesthesia,with 2×10⁶ live, wild-type CMS-5 cells in 0.5 ml HBSS. The groupsreceiving the HA-K-GM-CSF and control vaccines each consisted of 5 mice,whereas the group receiving the GM-CSF-K-HA vaccine consisted of 4 micebecause 1 mouse failed to awaken from anesthesia.

[0684] Tumor development was assessed daily by palpation and visualinspection. The observer was blinded to the vaccine received by each setof mice to ensure against bias. Mice were sacrificed by CO2 asphyxiationwhen tumors become unwieldy. All mice that had received the controlvaccine developed tumors within 18 days after challenge with live tumorcells. In contrast, 100% of mice that had received the GM-CSF-K-HAvaccine and 60% of mice that had received the HA-K-GM-CSF vaccineremained tumor-free at the end of the experiment, 40 days afterchallenge. Thus, immunization with a composition comprising tumor cellsand a molecule of the invention confers significantly longer tumor-freesurvival than immunization with a composition comprising tumor cells butnot comprising a molecule of the invention.

Example 17

[0685] Cloning of Human GM-CSF-K-HA

[0686] pUC19 human GM-CSF-K-HA (hGM-CSF-K-HA) is cloned starting withpUC19 GM-CSF-K-HA. pUC19 GM-CSF-K-HA. is digested with EcoRI and NgoMIV. EcoRI cuts at the 5′ end of the murine GM-CSF coding sequence andNgo M IV cuts at the 3′ end of the murine GM-CSF molecule. The resultingplasmid with the murine GM-CSF coding region removed is purified afterelectrophoresis through agarose gel using a kit manufactured by Qiagen.The human GM-CSF coding segment is generated by PCR from a commerciallyavailable human cDNA library (Clontech). The human sequence begins atamino acid 18, the start of the mature protein, i.e. lacking thesecretory signal sequence. The 3′ end corresponds to amino acid 144,eliminating the endogenous termination codon. Upstream hGM-CSF Primer5′GCGAATTCCGGCCGGCACCCGCCCGCTCGCCCAGC Downstream hGM-CSF Primer5′TAGCCGGCCTCCTGGACTGGCTCCCAGCA

[0687] Conditions for PCR are: Denaturation 90° one minute Annealing 60°one minute Extension 72° one minute

[0688] PCR is performed for 20 cycles using vent polymerase.

[0689] Following PCR, the product is electrophoresed through a 1.0%agarose gel and the hGM-CSF gene is extracted from the gel using aQiagen kit according to the manufacturer's instructions. The purifiedhGM-CSF DNA fragment is digested with Eco RI and NgoM IV and ligatedinto the pUC19 murine GM-CSF-K-HA vector that has been digested withEcoRI and NgoM IV to remove the murine GM-CSF sequence. The DNA is usedto transform E.coli AG1 and transformants are selected on LB-ampicillinplates. Plasmid DNA from individual colonies is isolated and digestedwith restriction enzymes to identify clone harboring a pUC19hGM-CSF-K-HA plasmid.

[0690] The pUC19 hGM-CSF-K-HA plasmid is purified according to themanufacturer's instructions using a kit purchased from Qiagen. PCR ofpUC19 hGM-CSF-K-HA is used to generate a DNA fragment encodinghGM-CSF-K-HA for cloning into a yeast expression vector. The PCR productcontains Eag I cloning sites for in frame insertion into the yeastexpression vector. Upstream Primer 5′GCGAATTCCGGCCGGCACCCGCCCGCTCGCCCAGCDownstream Primer 5′ATGGTACCCGGCCGTTATCATCTGGATTGAATGGACGG

[0691] Conditions for PCR are: Denaturation 90° one minute Annealing 60°one minute Extension 72° one minute

[0692] PCR is performed for 20 cycles using vent polymerase.

[0693] Following PCR the product is electrophoresed through a 1.0%agarose gel and the hGM-CSF-K-HA gene is extracted from the gel using aQiagen kit according to the manufacturer's instructions. The purifiedDNA fragment is digested with Eag I and ligated to the yeast expressionvector ITK that has been digested with Eag I. The ITK vector is designedfor (1) replication in E.coli and (2) expression of genes in the yeastSaccharomyces cerevisiae after stable integration using homologousrecombination. The vector contains:

[0694] 1. Sequences for replication of the plasmid in E.coli

[0695] 2. Yeast Gal promoter for expression of heterologous genes inyeast grown in media containing galactose.

[0696] 3. PrePro—Synthetic DNA sequence, optimized for secretion andsignal sequence cleavage of distal genes in yeast.

[0697] 4. Unique Eag I site for cloning genes to be expressed.

[0698] 5. Alpha terminator—DNA sequence for efficient termination ofproximal genes.

[0699] 6. Delta sequence that allows for stable integration of theplasmid by recombination with endogenous delta sequences in the yeastchromosome.

[0700] 7. Antibiotic resistance gene allowing for selection in E.coliwith kanamycin and selection in yeast with G418.

[0701] This plasmid is used to transform E.coli strain AG-1.Transformants are selected by growth on LB plates containing 100 ug/mlkanamycin. Individual colonies are grown in LB media containingkanamycin and plasmids are purified. Restriction digests determineorientation of inserts. The resulting plasmid ITK hGM-CSF-K-HA ispurified using a kit purchased from Qiagen according to themanufacturer's instructions.

[0702] The purified plasmid is linearized with Mfe 1 and used totransform the yeast strain Saccharomyces cerevisiae WDHY131 usinglithium acetate (LiAc). A 10 ml culture of S.cerevisiae grown tosaturation at 30° in YPD media (per liter/20 g Bactotryptone; 20 gdextrose; 10 g yeast extract) is used to inoculate 100 ml of YPD. Theculture is grown at 30° with shaking for 3 hours. The yeast areharvested by centrifugation at 11,000×g for 2 minutes and resuspended in25 ml of sterile water. The yeast are centrifuged as above andresuspended in 1.0 ml of 100 mM lithium acetate and transferred to a 1.5ml microfuge tube. The yeast are pelleted by centrifugation at 12,000×gfor 15 seconds and the supernatant removed. The cells are resuspended in0.5 ml of 100 mM LiAc. 50 uL of cell suspension is added to individualmicrofuge tubes and centrifuged as above. Supernatant is removed.Transformation mix added to the yeast pellet consists of: 240 uL PEG(50% w/v); 36 uL 1.0 M LiAc; 5 uL single stranded DNA (10 mg/ml) and 1ug of linearized ITK hGM-CSF-K-HA in 75 uL of water. The mixture isvortexed to resuspend the cell pellet and incubated at 30° for 30minutes. The cells are then shocked at 42° for 15 minutes, centrifugedto pellet, and resuspended in 0.5 ml of YPD. Yeast are incubated in YPDmedia for 3 hours and plated on YPD plates containing 2 mg/ml G418.Plates are grown at 30° for 3 days until individual colonies appear.

[0703] To screen for expression of hGM-CSF-K-HA, individual colonies aregrown in 1 ml of YPD media at 30° for 2 days. The cells are centrifugedat 8,000×g for 2 minutes and the YPD media removed and replaced with 1ml of YPG media (per liter/20 g Bactotryptone; 20 g galactose; 10 gyeast extract) for induction from the gal promoter. Yeast are grown inYPG media for 2 days. An aliquot is then removed and the cells arepelleted. The supernatant is tested for hGM-CSF expression using anELISA kit purchased from Endogen. Protocol is according to themanufacturer.

1. A fusion polypeptide comprising a lectin which comprises at leastabout 10 contiguous amino acids of an influenza virus hemagglutinin; andat least about 5 contiguous amino acids of a naturally occurring GM-CSFmolecule.
 2. The fusion polypeptide of claim 1 in which said lectin isN-terminal to said contiguous amino acids of a naturally occurringGM-CSF molecule.
 3. The fusion polypeptide of claim 1 in which saidlectin is C-terminal to said contiguous amino acids of a naturallyoccurring GM-CSF molecule.
 4. The fusion polypeptide of claim 1 in whichsaid lectin comprises at least about 10 contiguous amino acids of theHA1 domain of an influenza virus hemagglutinin.
 5. The fusionpolypeptide of claim 4 which comprises the HA1 domain of an influenzavirus hemagglutinin.
 6. The fusion polypeptide of claim 1 in which saidinfluenza virus hemagglutinin is a hemagglutinin of an influenza Avirus.
 7. The fusion polypeptide of claim 1 in which said influenzavirus hemagglutinin is of a subtype from a virus that infects humans. 8.The fusion polypeptide of claim 1 in which said influenza virushemagglutinin is of an H1 subtype.
 9. The fusion polypeptide of claim 1in which said influenza virus hemagglutinin is from the influenza Astrain PR/8/34.
 10. The fusion polypeptide of claim 1 in which saidinfluenza virus hemagglutinin is of an H2 subtype.
 11. The fusionpolypeptide of claim 1 in which said influenza virus hemagglutinin is ofan H3 subtype.
 12. The fusion polypeptide of claim 1 in which saidinfluenza virus hemagglutinin is of a subtype from a virus that does notinfect humans.
 13. The fusion polypeptide of claim 1 which comprises theentire amino acid sequence of a naturally occurring GM-CSF molecule. 14.The fusion polypeptide of claim 1 in which said GM-CSF molecule is amurine GM-CSF.
 15. The fusion polypeptide of claim 1 in which saidGM-CSF molecule is a human GM-CSF.